Methods of treating autoimmune disease or chronic inflammation with antibodies that bind and block triggering receptor expressed on myeloid cells-1 (trem-1)

ABSTRACT

The invention relates to antibodies that are capable of specifically binding TREM-1 and preventing the activation of TREM-1, a protein expressed on monocytes, macrophages and neutrophils. Such antibodies find utility in the treatment of individuals with an inflammatory disease, such as rheumatoid arthritis and inflammatory bowel disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/209,627, filed Dec. 4, 2018; which is a divisional of U.S.application Ser. No. 15/372,600, filed Dec. 8, 2016, issued as U.S. Pat.No. 10,189,904, which is a continuation of US application Ser. No.13/954,318, filed Jul. 30, 2013, issued as U.S. Pat. No. 9,550,830;which is a continuation-in-part of US application Ser. No. 13/690,748,filed Nov. 30, 2012, issued as U.S. Pat. No. 9,000,127; which claimedpriority of European Patent Application No. 12176892.3, filed Jul. 18,2012, European Patent Application No. 12158974.1, filed Mar. 12, 2012,and European Patent Application No. 12155641.9, filed Feb. 15, 2012; andthis application also claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/674,434, filed Jul. 23, 2012, U.S.Provisional Application No. 61/672,799, filed Jul. 18, 2012, U.S.Provisional Application No. 61/599,447, filed Feb. 16, 2012, and U.S.Provisional Application No. 61/598,968, filed Feb. 15, 2012. Thecontents of all above-named applications are incorporated herein byreference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 105218-03-5004-US-05_Sequence_Listing_ST25; Size: 40KB; and Date of Creation: Jan. 31, 2021) is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of identifying a functionalTREM-1 antibody. The invention also relates to antibodies that arecapable of specifically binding TREM-1 and which are capable of reducingor blocking TREM-1 activity (signalling and/or activation). Furthermore,the invention relates to uses for such antibodies, such as therapeuticand pharmaceutical uses.

BACKGROUND OF THE INVENTION

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a receptorthat is expressed on monocytes, macrophages and neutrophils. Whenactivated, TREM-1 associates with a signalling protein, DAP12, andtriggers the release of pro-inflammatory cytokines from the cells thatexpress it (Bouchon et al, J. Immunol. 2000; 164(10): 4991-4995). TREM-1mRNA and protein expression is known to be upregulated in the myeloidcells of individuals with sepsis, rheumatoid arthritis (RA) andinflammatory bowel disease (IBD). Increasing scientific evidencesupports the theory that TREM-1 contributes to the development andprogression of inflammatory diseases, as TREM-1 positive monocytes andneutrophils that are recruited to an inflamed area exacerbateinflammation (Bouchon et al. Nature 2001: 410: 1103-1107; Schenk et al,Clin Invest. 2007; 117(10): 3097-3106); Kuai et al., Rheumatology(Oxford). 2009; 48(11)-1352-1358.

Antibodies that are capable of binding TREM-1 are known, including thecommercially available TREM26 and TREM37 (cat. nos. 314902 and 316102,respectively, Biolegend, San Diego, Calif. 92121, USA), MAB1278 (cat.no. MAB1278, R&D Systems. Minneapolis, Minn. 55413, USA), mAb 6B1 (cat.no. HM2252, Hycult Biotech, Uden, Netherlands) and anti-TREM-1 2E2 (cat.no. HPA005563, Sigma-Aldrich, Denmark). All known TREM-1 antibodies areagonistic when immobilised; that is, they increase cytokine release frommonocytes, macrophages and neutrophils. Another characteristic of theknown TREM-1 antibodies is that they do not cross-react with TREM-1 fromprimates, such as cynomolgus monkeys or rhesus monkeys, which means thatthe known antibodies cannot be tested in these animals.

Thus, there is a need in the an for an antibody that is capable ofbinding and blocking the function of TREM-1. There is a need in the artfor a TREM-1 antibody that is capable of preventing TREM-1 from formingdimers/multimers. There is a need in the art for a TREM-1 antibody thatis capable of blocking TREM-1 activation and signalling. There is a needin the art for a TREM-1 antibody that is capable of interfering with theinteraction between TREM-1 and its ligand. There is a need in the artfor a TREM-1 antibody that is capable of blocking cytokine release froma myeloid cell. There is a need in the art for a TREM-1 antibody thathas little or no agonistic activity when soluble or immobilised. Thereis also a need in the art for an antibody that is capable of bindingboth human TREM-1 and TREM-1 from one or more other species, such as aprimate, in order to enable toxicology investigation as well as assessthe pharmacokinetics and pharmacodynamics of the antibody in suitableanimal models.

Disclosed herein are TREM-1 antibodies that are suitable for use aspharmaceuticals. Such antibodies may have a substantial impact upon thequality of life of individuals with sepsis or a chronic inflammatorydisease such as rheumatoid arthritis, psoriatic arthritis andinflammatory bowel disease.

SUMMARY

The present invention relates to a method of identifying a functionalTREM-1 antibody, comprising (a) culturing a first cell expressingTREM-1, a signalling protein and a reporter construct; (b) measuring theactivity of the first cell when said cell is incubated with a TREM-1modifying agent, (c) contacting the co-culture of (b) with a TREM-1antibody; and (d) measuring that the activity of the first cell is lessthan or more than the activity measured in (b).

The method may be tailored to identify a blocking TREM-1 molecule, suchas an antibody. The method of identifying a blocking TREM-1 antibodycomprises (a) culturing a first cell expressing TREM-1, a signallingprotein and a reporter construct; (b) measuring the activity of thefirst cell when said cell is incubated with an activating compound, suchas a TREM-1 ligand, or an activated neutrophil; (c) contacting theco-culture of the first cell and the activating compound, such as aTREM-1 ligand, or an activated neutrophil with a TREM-1 antibody; and(d) measuring that the activity of the first cell is less than theactivity measured in (b).

The method may be tailored to identify a stimulating TREM-1 antibody.The method of identifying a stimulating TREM-1 antibody comprises (a)culturing a first cell expressing TREM-1, a signalling protein and areporter construct; (b) measuring the activity of the first cell; (c)contacting/incubating said cell with a TREM-1 antibody; and (d)measuring that the activity of the first cell is more than the activityof the measured in (b).

The first cell may be of haematopoetic origin. The modifying agent of(b) may be an activated neutrophil or a TREM-1 ligand. The signallingprotein may be DAP10, DAP12, TCR zeta. Fc gamma RIII and an Fc receptor,or a portion thereof. The signalling protein may signal via atranscription factor such as NFAT or NFκB. The reporter gene may be agene that is not natively expressed in said first cell and may be a genethat encodes β-galactosidase, luciferase, green fluorescent protein(CFP) or chloramphenicol transferase. The present invention also relatesto stimulating TREM-1 antibodies that may be identified by means of theinvented method.

The present invention relates to antibodies that are capable ofspecifically binding to TREM-1 and that are capable of blocking TREM-1function. Said antibodies may be capable of preventing or reducing thedimerisation or multimerisation of TREM-1. Said antibodies may becapable of blocking the interaction between TREM-1 and its ligand, orthe antibodies may be capable of blocking the TREM-1 function that isinduced by a TREM-1 ligand. The TREM-1 may be human TREM-1 and/or TREM-1from another species than a human, such as TREM-1 from another primatethan a human.

Antibodies of the invention may be capable of competing with mAb 0170for binding to human TREM-1. Antibodies of the invention may be capableof specifically binding a polypeptide comprising amino acids D38 to F48of SEQ ID NO: 1 (human TREM-1). Antibodies of the invention may have anepitope comprising one, two, three, four, five, six, seven or all of theamino acid residues selected from the group consisting of the D38, V39,K40, C41, D42, Y43, T44 and L45 of SEQ ID NO: 1 (human TREM-1) and one,two or all of the amino acid residues selected from the group consistingof the E46, K47 and F48 of SEQ ID NO: 1 (human TREM-1), as may bedetermined using HX-MS. Antibodies of the invention may have an epitopecomprising one, two, three or all of the amino acid residues selectedfrom the group consisting of the D42, E46, D92 and H93 of SEQ ID NO: 1(human TREM-1), as may be determined by measuring antibody binding tovariants of TREM-1.

Antibodies of the invention may be capable of competing with mAb 0170for binding to cynomolgus monkey TREM-1. Antibodies of the invention maybe capable of capable of specifically binding a polypeptide comprisingamino acids E19 to L26 of cynomolgus monkey TREM-1 (SEQ ID NO: 12), orthe corresponding amino acids of SEQ ID NO: 21, as may be determinedusing HX-MS.

Antibodies of the invention may be used as pharmaceuticals for thetreatment of individuals with one or more autoimmune diseases and/orchronic inflammation. Hence, the present invention also relates to amethod of treatment of individuals with one or more autoimmune diseasesand/or chronic inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Activation of the BWZ.36/hTREM-1:DAP2 NFAT-LacZ cell line (hereinalso referred to as the “BWZ/hTREM-1 reporter cell”) is reduced byantibodies such as 14F128 and 14F113 but not by the commerciallyavailable antibodies MAB1278 MAB1278 (R&D Systems, Minneapolis, Minn.55413, USA: Cat. No. MAB1278), anti-TREM-1 HPA (Sigma, USA: Cat. No.HPA005563), aTREM26 (Biolegend, San Diego, Calif. 92121, USA: Cat. No.314902) and aTREM37 (Biolegend, San Diego, Calif. 92121, USA. Cat. No.316102).

FIG. 2: The BWZ/hTREM-1 reporter cell alone or co-cultured with TLRLpre-stimulated neutrophils. TLRL activated neutrophils are able toinduce a 15-fold increase in the signal (luminescence), compared tonon-activated neutrophils (see the last two columns). The positivecontrol (the platebound, agonistic MAB1278 (R&D Systems, Minneapolis,Minn. 55413, USA: Cat. no. MAB1278) gave a 32-fold induction in thisexperiment (see second column). Data are plotted as means±SEM (n=3).

FIG. 3: A normalised reporter assay, in which TREM-1 is stimulated byPGN-activated neutrophils, shows that the commercially availableantibodies TREM-26 (cat. no. 314902, Biolegend, San Diego, Calif. 92121,USA), TREM-37 (cat. no. 316102, Biolegend, San Diego, Calif. 92121,USA), MAB1278 (cat. no. MAB1278, R&D Systems, Minneapolis, Minn. 55413,USA), and anti-TREM-1 HPA (cat. no. HPA005563, Sigma, St Louis, Mo.,USA) are agonistic antibodies that enhance the TREM-1 dependentluminescence signal in the reporter assay (giving a signal higher than1). The antibody identified as 5F27A1 and 14F69 are shown to be the bestblocker (giving a signal less than 0.5). Data plotted as mean±SEM (n=3).Isotype control MAB002 (cat. no. MAB002, R&D Systems, Minneapolis, Minn.55413, USA). All antibodies have been used at 1 μg/ml.

FIG. 4: Activity in the BWZ/hTREM-1 reporter assay. On the x-axis,TREM-1 blocking activity is shown as the activity that remained when 0.2μg/ml antibody was added to the BWZ/hTREM-1 reporter cell, which hadbeen pre-activated with PGN-stimulated neutrophils. When TREM-1antibodies were platebound, some (including all of the commerciallyavailable TREM-1 antibodies) were able to crossbind TREM-1 and therebyactivate TREM-1. This activity is shown on the y-axis. Thus, theantibodies shown as dots in the box in the lower left-hand corner allshow different properties compared to known antibodies, beingadvantageously blocking and not agonistic.

FIG. 5: Binding of human TREM-1 antibodies to PBMCs from a rhesusmonkey. In the upper left corner, it is shown that most of theantibodies only bound human TREM-1 (each black dot represents oneantibody). Some antibodies, such as mAbs 0025 and 14F128, 14F11, 14F29,14F113 and 14F69, were able to bind to both human TREM-1 and rhesusmonkey TREM-1, showing cross-reactivity.

FIGS. 6A-6C: Binding of anti-human TREM-1 antibodies to PBMCs from acynomolgus monkey. FIG. 6A: In the upper left corner, it is shown thatmost antibodies only bound human TREM-1 (each black dot represents oneantibody). Some antibodies, such as mAb 0025 and 14F128, 14F11, 14F29,were able to bind both human and cynomolgus monkey TREM-1, showingcross-reactivity. Some antibodies, such as 14F69 (mAb 0044), are able tobind cynomolgus monkey PBMCs (FIG. 6B) and human PBMCs (FIG. 6C) equallywell.

FIGS. 7A-7D: Sequence coverage of HX analyzed peptides of hTREM-1(residues 21-200 of SEQ ID NO:1) in the presence and absence of mAb 0023or mAb 0026 (FIG. 7A), mAb 0024 or Biolegend Clone 26 (FIG. 7B), mAb0025 (FIG. 7C) or RnD Biosystems' MAB1278 (FIG. 7D). The primarysequence is displayed above the HX analyzed peptides (shown ashorizontal bars). Peptides showing similar exchange patterns both in thepresence and absence of mAbs are displayed in white, whereas peptidesshowing reduced deuterium incorporation upon mAb binding are colouredblack. Boxed sequence regions define the epitope.

FIG. 8: Structural mapping of the epitope of mAb 0025 on hTREM-1. Thestructure of hTREM-1 (pdb 1Q8M) is represented with one part of thedimer as black C-alpha wire. The other part of the hTREM-1 dimer isshown in grey ribbon with the 0025 epitope displayed in black.

FIG. 9: The BWZ/hTREM-1 reporter cell co-cultured with TREM-1 ligandcomplex reflects the TREM-1 functionality which was dose-dependentlyinhibited by TREM-1 mAb-0170.

FIG. 10: TNFalpha release from M2 macrophages stimulated by PGLYRP-1 wasblocked by TREM-1 antibodies.

FIG. 11: The human TREM-1 (residues 17-200 of SEQ ID NO:1) andcynomolgus monkey TREM-1 (residues 2-180 of SEQ ID NO:12) sequences arealigned. Amino acid residues that differ between the two are shown inbold type. Residues that have been shown to be within the epitope (asdetermined using HX-MS and surface plasmon resonance) are highlighted.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 represents the amino acid sequence of wild type (wt) humanTREM-1.

SEQ ID NO: 2 represents the amino acid sequence of the variable heavychain of the m14F69 antibody.

SEQ ID NO: 3 represents the amino acid sequence of the variable lightchain of the m14F69 antibody.

SEQ ID NO: 4 represents the amino acid sequence of the heavy chain of afirst humanised TREM-1 antibody (mAb 0170).

SEQ ID NO: 5 represents the amino acid sequence of the light chain of afirst humanised TREM-1 antibody (mAB 0170).

SEQ ID NO: 6 represents the amino acid sequence of the heavy chain of asecond humanised TREM-1 antibody (mAb 0122).

SEQ ID NO: 7 represents the amino acid sequence of the light chain of asecond humanised TREM-1 antibody (mAb 0122).

SEQ ID NO: 8 represents the amino acid sequence of the heavy chain ofthe m14F128 antibody.

SEQ ID NO: 9 represents the amino acid sequence of the light chain ofthe m14F128 antibody.

SEQ ID NO: 10 represents the amino acid sequence of the heavy chain ofthe m14F113 antibody.

SEQ ID NO: 11 represents the amino acid sequence of the light chain ofthe m14F113 antibody.

SEQ ID NO: 12 represents the amino acid sequence of the extracellulardomain of wild type (wt) cynomolgus monkey (c) TREM-1, when expressed inE. coli.

SEQ ID NO: 13 represents the amino acid sequence ofK20A-hTREM-1-Cmyc2-His6 (construct 0222).

SEQ ID NO: 14 represents the amino acid sequence ofA24T/Y28F/N30S/R32Q/P70H-cTREM-1-Cmyc2-His6 (construct 0244).

SEQ ID NO: 15 represents the amino acid sequence ofA24T/Y28F/N30S/R32Q/ES4K-cTREM-1-Cmyc2-His6 (construct 0245).

SEQ ID NO: 16 represents the nucleic acid sequence of a primer

SEQ ID NO: 17 represents the nucleic acid sequence of a primer.

SEQ ID NO: 18 represents the amino acid sequence of human (h)TREM-1(1-134)-His6.

SEQ ID NO: 19 represents the amino acid sequence of cTREM-1-Cmyc2-His6(construct 0238).

SEQ ID NO: 20 represents the amino acid sequence of hTREM-1-Cmyc2-His6(construct 0247).

SEQ ID NO: 21 represents the amino acid sequence of full length cTREM-1.

SEQ ID NO: 22 represents the amino acid sequence of full length murine(m) TREM-1.

SEQ ID NO: 23 represents the amino acid sequence of full lengthhPGLYRP1.

SEQ ID NO: 24 represents the amino acid sequence of the extracellularIgV-like domain of human TREM-1, Met-human (h) TREM-1 (21-134).

SEQ ID NO: 25 represents the amino acid sequence of the heavy chain ofthe Fab region of monoclonal antibody (mAb)0170.

SEQ ID NO: 26 represents the amino acid sequence of the light chain ofthe Fab region of monoclonal antibody (mAb)0170.

DESCRIPTION

TREM-1 is a transmembrane protein that consists of 234 amino acids,including a single extracellular immunoglobulin domain and a shortcytoplasmic tail with no apparent signaling motif. When activated,TREM-1 associates with the ITAM-containing signaling adaptor protein,DAP12. Downstream signalling may include activation of the NFATtranscription factor, causing an upregulation of pro-inflammatorycytokine production.

The present invention relates to antibodies that are capable ofspecifically binding and blocking the function of TREM-1. Antibodies ofthe invention may block TREM-1 function by reducing/blocking TREM-1activation and downstream signalling.

Antibodies according to the invention may block TREM-1 by means of oneof one or a combination of several different mechanisms, blocking TREM-1directly or indirectly. For example, antibodies of the invention mayprevent the natural ligand of TREM-1, peptidoglycan recognition protein1 (PGLYRP1), from creating a functional complex with TREM-1 and/orantibodies of the invention may block TREM-1 by preventing individualTREM-1 molecules from forming dimers or multimers. TREM-1 dimerisationor multimerisation may be reduced or prevented by TREM-1 antibodies thatare capable of binding to a portion of TREM-1 that would otherwisereside in the interface of a TREM-1 dimer, thus preventing individualTREM-1 molecules from associating with one another. TREM-1 dimerisationor multimerisation may be reduced or prevented by TREM-1 antibodies thatinterfere with the interaction of TREM-1 with its ligand. Antibodiesaccording to the current invention may block PGLYRP1-induced activationof TREM-1. PGLYRP1, a highly conserved, 196 amino acid long proteinconsisting of a signal peptide and a peptidoglycan binding domain, isexpressed in neutrophils and released upon their activation. Antibodiesaccording to the current invention may down-regulate pro-inflammatorycytokine release from myeloid cells. Antibodies according to the currentinvention may block the release of TNFalpha, MIP-1beta, MCP-1, IL-1beta,GM.CSF, IL-6 and/or IL-8 from macrophages, neutrophils, synovial tissuecells and/or a reporter cell, as disclosed herein.

Antibodies of the invention may be capable of binding both human TREM-1and TREM-1 from another species than a human being. The term “TREM-1”,as used herein, thus encompasses any naturally occurring form of TREM-1which may be derived from any suitable organism. For example, TREM-1 foruse as described herein may be vertebrate TREM-1, such as mammalianTREM-1, such as TREM-1 from a primate (such as a human, a chimpanzee, acynomolgus monkey or a rhesus monkey); a rodent (such as a mouse or arat), a lagomorph (such as a rabbit), or an artiodactyl (such a cow,sheep, pig or camel). Preferably, the TREM-1 is SEQ ID NO: 1 (humanTREM-1). The TREM-1 may be a mature form of TREM-1 such as a TREM-1protein that has undergone post-translational processing within asuitable cell. Such a mature TREM-1 protein may, for example, beglycosylated. The TREM-1 may be a full length TREM-1 protein.

Antibodies of the invention may be monoclonal antibodies, in the sensethat they are directly or indirectly derived from a single clone of a Blymphocyte. TREM-1 antibodies may be produced, screened and purifiedusing, for example, the methods described in the Examples. In brief, asuitable mouse such as a TREM-1 or TREM-1REM-3 knock-out (KO) mouse maybe immunised with TREM-1, a cell expressing TREM-1 or a combination ofboth.

Antibodies of the invention may be polyclonal in the sense of being amixture of monoclonal antibodies according to the current invention.

Primary screening of hybridoma supernatants may be performed usingdirect ELISA or FMAT and secondary screening may be performed using flowcytometry. Positive hybridoma supernatants may then be screened in areporter gene assay.

Antibodies may be recombinantly expressed in prokaryotic or eukaryoticcells. The prokaryotic cell may be E. coli. The eukaryotic cell may be ayeast, insect or mammalian cell, such as a cell derived from an organismthat is a primate (such as a human, a chimpanzee, a cynomolgus monkey ora rhesus monkey), a rodent (such as a mouse or a rat), a lagomorph (suchas a rabbit) or an artiodactyl (such a cow, sheep, pig or camel).Suitable mammalian cell lines include, but are not limited to, HEK293cells, CHO cells and HELA cells. TREM-1 antibodies may also be producedby means of other methods known to the person skilled in the art, suchas a phage display or a yeast display.

Once produced, antibodies may be screened for binding to, for example,full length TREM-1 or mutants thereof using the methods described in theExamples.

One embodiment of the current invention is a method of identifying afunctional TREM-1 antibody. Antibodies that are capable of specificallybinding TREM-1 and that have any effect upon TREM-1 activation anddownstream signalling are herein referred to as “functional TREM-1antibodies”. A “functional” TREM-1 antibody herein refers to an antibodythat is capable of blocking or stimulating TREM-1. The method ofidentifying a functional TREM-1 antibody comprises (a) culturing a firstcell expressing TREM-1, a signalling protein and a reporter construct;(b) measuring the activity of the first cell when said cell is incubatedwith a TREM-1 modifying agent; (c) contacting the co-culture of (b) witha TREM-1 antibody; and (d) measuring that the activity of the first cellis less than or more than the activity measured in (b).

The “first cell” of (a) may be a cell of haematopoetic origin, such as amyeloid cell, such as a T-cell. The signalling protein of (a) may be anysignalling protein that is capable of forming a complex with TREM-1.Suitable signalling proteins include DAP10, DAP12, TCR zeta, Fc gammaRIII and an Fc receptor, or part thereof. The reporter construct of (a)may be any construct that is capable of being activated via thesignalling protein and generating a recognisable signal. Suitablereporter constructs comprise a transcription factor and a reporter gene.The signalling protein may signal via a transcription factor selectedfrom the group consisting of the NFAT and NFkB. The reporter gene is agene that is not natively expressed in said first cell and may be a genethat encodes β-galactosidase, luciferase, green fluorescent protein(GFP) or chloramphenicol transferase. Said first cell may be transfectedwith a transcription factor and a reporter gene using methods that arewell known in the art.

The “BWZ/hTREM-1 reporter cell” described in the Examples is one exampleof a “first cell”.

The modifying agent of (b) may be a TREM-1 ligand or an activatedneutrophil. The “TREM-1 antibody” of (c) may be a TREM-1 specifichybridoma supernatant or a purified antibody. The activity measured in(d) is the signal produced by the reporter construct. An example of suchsignalling is the luminescence caused by NFAT-driven LacZ β-lactamaseluciferase) production.

The method may be tailored to identify a blocking TREM-1 antibody. Themethod of identifying a blocking TREM-1 antibody comprises (a) culturinga first cell expressing TREM-1, a signalling protein and a reporterconstruct; (b) measuring the activity of the first cell when said cellis incubated with an activated neutrophil; (c) contacting the co-cultureof the first cell and the activated neutrophil with a TREM-1 antibody;and (d) measuring that the activity of the first cell is less than theactivity measured in (b).

The method may also be tailored to identify a stimulating TREM-1antibody. The method of identifying a stimulating TREM-1 antibodycomprises (a) culturing a first cell expressing TREM-1, a signallingprotein and a reporter construct; (b) measuring the activity of thefirst cell; (c) contacting/incubating said cell with a TREM-1 antibody,and (d) measuring that the activity of the first cell is more than theactivity of the measured in (b).

The present invention relates to blocking TREM-1 antibodies that may beidentified by means of the method, herein disclosed, of identifying ablocking antibody. When tested using the method described above and inthe Examples, an antibody according to the current invention may, at aconcentration of less than 100 μg/ml—such as less than 90 μg/ml, such asless than 80 μg/ml, such as less than 70 μg/ml, such as less than 60μg/ml, such as less than 50 μg/ml, such as less than 40 μg/ml, such asless than 30 μg/ml, such as less than 20 μg/ml, such as less than 10μg/ml, such as less than 5 μg/ml, such as less than 1 μg/ml—be capableof reducing the activity of said first cell by 50%, such as 60%, such as70%, such as 80%, such as 90%, such as 95%, such as 100%. An antibodyaccording to the invention may be capable of completely extinguishingthe activity of the first cell. When tested using the method describedabove and in the Examples, an antibody according to the currentinvention may, at a concentration of less than 1 μg/ml—such as less than0.9 μg/ml, such as less than 0.8 μg/m, such as less than 0.7 μg/ml, suchas less than 0.6 μg/ml, such as less than 0.5 μg/ml, such as less than0.4 μg/ml, such as less than 0.3 μg/ml, such as less than 0.2 μg/ml—becapable of extinguishing the activity of the first cell.

The present invention also relates to blocking TREM-1 antibodies thatmay be identified by other means than the method herein disclosed.

The term “antibody” herein refers to a protein, derived from a germlineimmunoglobulin sequence, which is capable of specifically binding to anantigen (TREM-1) or a portion thereof. The term includes full lengthantibodies of any class or isotype (that is, IgA, IgE, IgG, IgM and/orIgY) and any single chain or fragment thereof. An antibody thatspecifically binds to an antigen, or portion thereof, may bindexclusively to that antigen, or portion thereof, or it may bind to alimited number of homologous antigens, or portions thereof. Full-lengthantibodies usually comprise at least four polypeptide chains: two heavy(H) chains and two light (L) chains that are interconnected by disulfidebonds. One immunoglobulin sub-class of particular pharmaceuticalinterest is the IgG family. In humans, the IgG class may be sub-dividedinto 4 sub-classes: IgG1, IgG2, IgG3 and IgG4, based on the sequence oftheir heavy chain constant regions. The light chains can be divided intotwo types, kappa and lambda, based on differences in their sequencecomposition. IgG molecules are composed of two heavy chains, interlinkedby two or more disulfide bonds, and two light chains, each attached to aheavy chain by a disulfide bond. A heavy chain may comprise a heavychain variable region (VH) and up to three heavy chain constant (CH)regions: CH1, CH2 and CH3. A light chain may comprise a light chainvariable region (VL) and a light chain constant region (CL). VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). VH andVL regions are typically composed of three CDRs and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order: FR1,CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavyand light chains form a [binding] domain that is capable of interactingwith an antigen, whilst the constant region of an antibody may mediatebinding of the immunoglobulin to host tissues or factors, including butnot limited to various cells of the immune system (effector cells), Fcreceptors and the first component (Clq) of the classical complementsystem.

Antibodies of the current invention may be isolated. The term “isolatedantibody” refers to an antibody that has been separated and/or recoveredfrom (an) other component(s) in the environment in which it was producedand/or that has been purified from a mixture of components present inthe environment in which it was produced.

Certain antigen-binding fragments of antibodies may be suitable in thecontext of the current invention, as it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. The term “antigen-binding fragment” of anantibody refers to one or more fragment(s) of an antibody that retainthe ability to specifically bind to an antigen, such as TREM-1, asdescribed herein. Examples of antigen-binding fragments include Fab,Fab′, F(ab)2, F(ab′)2, F(ab)S, Fv (typically the VL and VH domains of asingle arm of an antibody), single-chain Fv (scFv; see e.g., Bird etal., Science 1988; 242:42S-426; and Huston et al. PNAS 1988;85:5879-5883), dsFv, Fd (typically the VH and CHI domain), and dAb(typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains,monovalent molecules comprising a single VH and a single VL chain;minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see,e.g., Ill et al., Protein Eng 1997; 10:949-57); camel IgG; IgNAR; aswell as one or more isolated CDRs or a functional paratope, where theisolated CDRs or antigen-binding residues or polypeptides can beassociated or linked together so as to form a functional antibodyfragment. Various types of antibody fragments have been described orreviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005;2S:1126-1136; WO2005040219, and published U.S. Patent Applications20050238646 and 20020161201. These antibody fragments may be obtainedusing conventional techniques known to those of skill in the art, andthe fragments may be screened for utility in the same manner as intactantibodies.

An antibody of the invention may be a human antibody or a humanisedantibody. The term “human antibody”, as used herein, is intended toinclude antibodies having variable regions in which at least a portionof a framework region and/or at least a portion of a CDR region arederived from human germline immunoglobulin sequences. (For example, ahuman antibody may have variable regions in which both the framework andCDR regions are derived from human germline immunoglobulin sequences.)Furthermore, if the antibody contains a constant region, the constantregion is also derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g. mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo).

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma which includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

Human antibodies may be isolated from sequence libraries built onselections of human germline sequences, further diversified with naturaland synthetic sequence diversity.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “human antibody derivative” refers to any modified form of thehuman antibody, such as a conjugate of the antibody and another agent orantibody.

The term “humanised antibody”, as used herein, refers to ahuman/non-human chimeric antibody that contains one or more sequences(CDR regions or parts thereof) that are derived from a non-humanimmunoglobulin. A humanised antibody is, thus, a human immunoglobulin(recipient antibody) in which at least residues from a hyper-variableregion of the recipient are replaced by residues from a hyper-variableregion of an antibody from a non-human species (donor antibody) such asfrom a mouse, rat, rabbit or non-human primate, which have the desiredspecificity, affinity, sequence composition and functionality. In someinstances. FR residues of the human immunoglobulin are replaced bycorresponding non-human residues. An example of such a modification isthe introduction of one or more so-called back-mutations, which aretypically amino acid residues derived from the donor antibody.Humanisation of an antibody may be carried out using recombinanttechniques known to the person skilled in the art (see, e.g., AntibodyEngineering, Methods in Molecular Biology, vol. 248, edited by Benny K.C. Lo). A suitable human recipient framework for both the light andheavy chain variable domain may be identified by, for example, sequenceor structural homology. Alternatively, fixed recipient frameworks may beused, e.g., based on knowledge of structure, biophysical and biochemicalproperties. The recipient frameworks can be germline derived or derivedfrom a mature antibody sequence CDR regions from the donor antibody canbe transferred by CDR grafting. The CDR grafted humanised antibody canbe further optimised for e.g. affinity, functionality and biophysicalproperties by identification of critical framework positions wherere-introduction (backmutation) of the amino acid residue from the donorantibody has beneficial impact on the properties of the humanisedantibody. In addition to donor antibody derived backmutations, thehumanised antibody can be engineered by introduction of germlineresidues in the CDR or framework regions, elimination of immunogenicepitopes, site-directed mutagenesis, affinity maturation, etc.

Furthermore, humanised antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, a humanised antibody will comprise at least one—typicallytwo—variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and in whichall or substantially all of the FR residues are those of a humanimmunoglobulin sequence. The humanised antibody can, optionally, alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin.

The term “humanised antibody derivative” refers to any modified form ofthe humanised antibody, such as a conjugate of the antibody and anotheragent or antibody.

The term “chimeric antibody”, as used herein, refers to an antibodywhose light and heavy chain genes have been constructed, typically bygenetic engineering, from immunoglobulin variable and constant regiongenes that originate from different species. For example, the variablesegments of genes from a mouse monoclonal antibody may be joined tohuman constant segments.

The fragment crystallisable region (“Fc region”/“Fc domain”) of anantibody is the N-terminal region of an antibody, which comprises theconstant CH2 and CH3 domains. The Fc domain may interact with cellsurface receptors called Fc receptors, as well as some proteins of thecomplement system. The Fc region enables antibodies to interact with theimmune system. In one aspect of the invention, antibodies may beengineered to include modifications within the Fc region, typically toalter one or more of its functional properties, such as serum half-life,complement fixation. Fc-receptor binding, protein stability and/orantigen-dependent cellular cytotoxicity, or lack thereof, among others.Furthermore, an antibody of the invention may be chemically modified(e.g., one or more chemical moieties can be attached to the antibody) orbe modified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. An IgG1 antibody may carry amodified Fc domain comprises one or more, and perhaps all of thefollowing mutations that will result in decreased affinity to certain Fcreceptors (L234A, L235E, and G237A) and in reduced C1q-mediatedcomplement fixation (A330S and P331S), respectively (residue numberingaccording to the EU index).

The isotype of an antibody of the invention may be IgG, such as IgG1,such as IgG2, such as IgG4. If desired, the class of an antibody may be“switched” by known techniques. For example, an antibody that wasoriginally produced as an IgM molecule may be class switched to an IgGantibody. Class switching techniques also may be used to convert one IgGsubclass to another, for example: from IgG1 to IgG2 or IgG4; from IgG2to IgG1 or IgG4; or from IgG4 to IgG1 or IgG2. Engineering of antibodiesto generate constant region chimeric molecules, by combination ofregions from different IgG subclasses, can also be performed.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further for instancein U.S. Pat. No. 5,677,425 by Bodmer et al.

The constant region may be modified to stabilize the antibody, e.g., toreduce the risk of a bivalent antibody separating into two monovalentVH-VL fragments. For example, in an IgG4 constant region, residue S228(residue numbering according to the EU index) may be mutated to aproline (P) residue to stabilise inter heavy chain disulphide bridgeformation at the hinge (see, e.g., Angal et al., Mol Immunol. 1995; 30:105-8).

Antibodies or fragments thereof may also be defined in terms of theircomplementarity-determining regions (CDRs). The term“complementarity-determining region” or “hypervariable region”, whenused herein, refers to the regions of an antibody in which amino acidresidues involved in antigen binding are situated. The region ofhypervariability or CDRs can be identified as the regions with thehighest variability in amino acid alignments of antibody variabledomains. Databases can be used for CDR identification such as the Kabatdatabase, the CDRs e.g. being defined as comprising amino acid residues24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light-chain variable domainand 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variabledomain; (Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest. Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242) Alternatively CDRs can be defined as thoseresidues from a “hypervariable loop” (residues 26-33 (L1), 50-52 (L2)and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55(H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia andLesk, J. Mol Biol 1987; 196: 901-917). Typically, the numbering of aminoacid residues in this region is performed by the method described inKabat et at, supra. Phrases such as “Kabat position”, “Kabat residue”,and “according to Kabat” herein refer to this numbering system for heavychain variable domains or light chain variable domains. Using the Kabatnumbering system, the actual linear amino acid sequence of a peptide maycontain fewer or additional amino acids corresponding to a shorteningof, or insertion into, a framework (FR) or CDR of the variable domain.For example, a heavy chain variable domain may include amino acidinsertions (residue 52a, 52b and 52c according to Kabat) after residue52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c,etc. according to Kabat) after heavy chain FR residue 82. The Kabatnumbering of residues may be determined for a given antibody byalignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence.

The term “framework region” or “FR” residues refer to those VH or VLamino acid residues that are not within the CDRs, as defined herein.

The m14F69 antibody has a variable heavy chain sequence as shown in SEQID NO: 2 and a variable light chain sequence as shown in SEQ ID NO: 3.An antibody of the invention may comprise this variable heavy chainsequence and/or this variable light chain sequence. The m14F69 antibodyhas the CDR sequences shown at amino acids 31 to 35, 50 to 68 and 101 to110 of SEQ ID NO: 2 and amino acids 24 to 38, 54 to 60 and 93 to 101 ofSEQ ID NO: 3. An antibody of the invention may comprise 1, 2, 3, 4, 5 orall 6 of these CDR sequences. An antibody of the invention may compriseamino acids 101 to 110 of SEQ ID NO: 2.

The heavy chain of an antibody according to the invention may comprise aCDR1 sequence of amino acids 31 to 35 (TYAMH) of SEQ ID NO: 2, whereinone of these amino acids may be substituted by a different amino acid.

The heavy chain of an antibody according to the invention may comprise aCDR2 sequence of amino acids 50 to 68 (RIRTKSSNYATYYADSVKD) of SEQ IDNO: 2, wherein one, two or three of these amino acids may be substitutedby a different amino acid.

The heavy chain of an antibody according to the invention may comprise aCDR3 sequence of amino acids 101 to 110 (DMGQRRQFAY) of SEQ ID NO: 2,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.

The light chain of an antibody according to the invention may comprise aCDR1 sequence of amino acids 24 to 38 (RASESVDTFDYSFLH) of SEQ ID NO: 3,wherein one, two or three of these amino acids may be substituted with adifferent amino acid.

The light chain of an antibody according to the invention may comprise aCDR2 sequence of amino acids 54 to 60 (RASNLES) of SEQ ID NO: 3, whereinone or two of these amino acids may be substituted with a differentamino acid.

The light chain of an antibody according to the invention may comprise aCDR3 sequence of amino acids 93 to 101 (QQSNEDPYT) of SEQ ID NO: 3,wherein one or two of these amino acids may be substituted with adifferent amino acid.

The mAb 0170 antibody has a heavy chain sequence as shown in SEQ ID NO:4 and a light chain sequence as shown in SEQ ID NO: 5. An antibody ofthe invention may comprise this heavy chain sequence and/or this lightchain sequence. The mAb 0170 antibody has the CDR sequences shown atamino acids 31 to 35, 50 to 68 and 101 to 110 of SEQ ID NO: 4 and aminoacids 24 to 38, 54 to 60 and 93 to 101 of SEQ ID NO. S. An antibody ofthe invention may comprise 1, 2, 3, 4, or all 6 of these CDR sequences.

The mAb 0122 antibody has a heavy chain sequence as shown in SEQ ID NO:6 and a light chain sequence as shown in SEQ ID NO: 7. An antibody ofthe invention may comprise this heavy chain sequence and/or this lightchain sequence. The mAb 0122 antibody has the CDR sequences shown atamino acids 31 to 35, 50 to 68 and 101 to 110 of SEQ ID NO: 6 and aminoacids 24 to 38, 54 to 60 and 93 to 101 of SEQ ID NO: 7. An antibody ofthe invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDRsequences.

The heavy chain of an antibody according to the invention may comprise aCDRH1 sequence of amino acids 31 to 35 (TYAMH) of SEQ ID NO: 4 or SEQ IDNO: 6, wherein one of these amino acids may be substituted by adifferent amino acid residue.

The heavy chain of an antibody according to the invention may comprise aCDRH2 sequence of amino acids 50 to 68 (RIRTKSSNYATYYAASVKG) of SEQ IDNO: 4 or SEQ ID NO: 6, wherein one, two or three of these amino acidsmay be substituted by a different amino acid.

The heavy chain of an antibody according to the invention may comprise aCDRH3 sequence of amino acids 101 to 110 (DMGIRRQFAY) of SEQ ID NO: 4,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.

The heavy chain of an antibody according to the invention may comprise aCDRH3 sequence of amino acids 101 to 110 (DMGQRRQFAY) of SEQ ID NO: 6,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.

The light chain of an antibody according to the invention may comprise aCDRL1 sequence of amino acids 24 to 38 (RASESVDTFDYSFLH) of SEQ ID NO: 5or SEQ ID NO: 7, wherein one, two or three of these amino acids may besubstituted with a different amino acid.

The light chain of an antibody according to the invention may comprise aCDRL2 sequence of amino acids 54 to 60 (RASNLES) of SEQ ID NO: 5 or SEQID NO: 7, wherein one or two of these amino acids may be substitutedwith a different amino acid.

The light chain of an antibody according to the invention may comprise aCDRL3 sequence of amino acids 93 to 101 (QQSNEDPYT) of SEQ ID NO: 5 orSEQ ID NO: 7, wherein one or two of these amino acids may be substitutedwith a different amino acid.

The m14F128 antibody has a heavy chain as shown in SEQ ID NO: 8 and alight chain as shown in SEQ ID NO: 9. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The m14F128 antibody has the CDR sequences shown atamino acids 31 to 35, 50 to 68 and 101 to 110 of SEQ ID NO: 8 and aminoacids 24 to 38, 54 to 60 and 93 to 101 of SEQ ID NO: 9. An antibody ofthe invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDRsequences.

The m14F113 antibody has a heavy chain as shown in SEQ ID NO. 10 and alight chain as shown in SEQ ID NO: 11. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The m14F113 antibody has the CDR sequences shown atamino acids 31 to 35, 50 to 68 and 101 to 110 of SEQ ID NO: 10 and aminoacids 24 to 38, 54 to 60 and 93 to 101 of SEQ ID NO: 11. An antibody ofthe invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDRsequences. An antibody of the invention may comprise amino acids 101 to110 of SEQ ID NO: 10.

The term “antigen” (Ag) refers to the molecular entity used forimmunization of an immunocompetent vertebrate to produce the antibody(Ab) that recognizes the Ag. Herein, Ag is termed more broadly and isgenerally intended to include target molecules that are specificallyrecognized by the Ab, thus including fragments or mimics of the moleculeused in the immunization process, or other process, e.g. phage display,used for generating the Ab.

The term “epitope”, as used herein, is defined in the context of amolecular interaction between an “antigen binding polypeptide”, such asan antibody (Ab), and its cot-responding antigen (Ag). Generally,“epitope” refers to the area or region on an Ag to which an Abspecifically binds, i.e. the area or region in physical contact with theAb. Physical contact may be defined through various criteria (e.g. adistance cut-off of 2-6 Å, such as 3 Å, such as 4 Å, such as 5 Å; orsolvent accessibility) for atoms in the Ab and Ag molecules. A proteinepitope may comprise amino acid residues in the Ag that are directlyinvolved in binding to a Ab (also called the immunodominant component ofthe epitope) and other amino acid residues, which are not directlyinvolved in binding, such as amino acid residues of the Ag which areeffectively blocked by the Ab, i.e. amino acid residues within the“solvent-excluded surface” and/or the “footprint” of the Ab.

The term epitope herein comprises both types of binding region in anyparticular region of TREM-1 that specifically binds to a TREM-1antibody. TREM-1 may comprise a number of different epitopes, which mayinclude, without limitation, conformational epitopes which consist ofone or more non-contiguous amino acids located near each other in themature TREM-1 conformation and post-translational epitopes whichconsist, either in whole or par, of molecular structures covalentlyattached to TREM-1, such as carbohydrate groups.

The epitope for a given antibody (Ab)/antigen (Ag) pair can be describedand characterized at different levels of detail using a variety ofexperimental and computational epitope mapping methods. The experimentalmethods include mutagenesis, X-ray crystallography, Nuclear MagneticResonance (NMR) spectroscopy, Hydrogen deuterium eXchange MassSpectrometry (HX-MS) and various competition binding methods; methodsthat are known in the art. As each method relies on a unique principle,the description of an epitope is intimately linked to the method bywhich it has been determined. Thus, depending on the epitope mappingmethod employed, the epitope for a given Ab/Ag pair may be describeddifferently.

At its most detailed level, the epitope for the interaction between theAg and the Ab can be described by the spatial coordinates defining theatomic contacts present in the Ag-Ab interaction, as well as informationabout their relative contributions to the binding thermodynamics. At aless detailed level, the epitope can be characterized by the spatialcoordinates defining the atomic contacts between the Ag and Ab. At aneven less detailed level the epitope can be characterized by the aminoacid residues that it comprises as defined by a specific criteria suchas the distance between or solvent accessibility of atoms in the Ab:Agcomplex. At a further less detailed level the epitope can becharacterized through function, e.g. by competition binding with otherAbs. The epitope can also be defined more generically as comprisingamino acid residues for which substitution by another amino acid willalter the characteristics of the interaction between the Ab and Ag.

In the context of an X-ray derived crystal structure defined by spatialcoordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag,the term epitope is herein, unless otherwise specified or contradictedby context, specifically defined as TREM-1 residues characterized byhaving a heavy atom (i.e. a non-hydrogen atom) within a distance of,eg., 4 Å from a heavy atom in the Ab.

From the fact that descriptions and definitions of epitopes, dependanton the epitope mapping method used, are obtained at different levels ofdetail, it follows that comparison of epitopes for different Abs on thesame Ag can similarly be conducted at different levels of detail.

Epitopes described on the amino acid level, e.g. determined from anX-ray structure, are said to be identical if they contain the same setof amino acid residues. Epitopes are said to overlap if at least oneamino acid is shared by the epitopes. Epitopes are said to be separate(unique) if no amino acid residue are shared by the epitopes.

Epitopes may also be defined indirectly, by means of comparing thebinding kinetics of antibodies to wild type human TREM-1 with those ofhuman TREM-1 variants that have alanine mutations in anticipatedepitopes. Decreased affinity or abrogated binding of an antibody tovariants of human TREM-1 in which an amino acid residue has beenreplaced with an alanine residue indicates that the mutated amino acidcontributes to the interaction between said antibody and wild type humanTREM-1. This approach provides a negative identification of the epitope.The method is compromised in effectively defining the epitope by thefact that protein misfolding or unfolding would give similar results asabrogation of interaction. The analysis can be complemented bycomparative gain of function mutational analyses of an orthologoustarget protein (eg., cynomolgus monkey TREM-1), if a cross-reactiveantibody exists. The comparison will define the epitope differencesbetween the antibody that does not cross-react with, eg., cynomolgusmonkey TREM-1 and the cross-reactive antibody.

Indirect identification of the epitope can also be provided by means ofmeasuring antibody (or antibody fragment) binding to variants of thewild type antigen (TREM-1). If an antibody or fragment thereof binds,eg., human but not cynomolgus monkey TREM-1 and if said antibody orfragment thereof is capable of binding a partly humanised variant ofcynomolgus monkey TREM-1 then this regained binding indicates that thesubstituted amino acid residue(s) is/are important for the interactionof the antibody with the antigen. In the same way, increased affinityfor humanized variants of cynomolgus monkey TREM-1, of an anti-humanTREM-1 antibody (or its Fab fragment) that has a weaker binding tocynomolgus monkey TREM-1 compared to human TREM-1, can provideinformation on the identity of residues composing the binding epitope.

The effect of the same mutations on any given cross-reactive antibodymakes it possible to discriminate between possible protein misfolding(abrogated binding to both antibodies) and loss of interaction in humanTREM-1 (binding to one of the antibodies and abrogated binding to theother antibody), whilst unambiguously providing information on theepitope differences between the antibody that does not cross-react andthe cross reactive antibody on an amino acid level.

Antibodies of the current invention may be capable of binding variantsof human TREM-1. Antibodies of the invention may be capable of bindingK20A-hTREM-1-Cmyc2-His6 (SEQ ID NO: 13), as determined using, eg.,surface plasmon resonance.

Antibodies of the current invention may be capable of binding variantsof cynomolgus monkey TREM-1. Antibodies of the invention may be capableof binding A24T/Y28F/N30S/R32Q/P70H-cTREM-1-Cmyc2-His6 (SEQ ID NO: 14),as determined using, eg., surface plasmon resonance. Antibodies of theinvention may be capable of bindingA24T/Y28F/N30S/R32Q/E54K-cTREM-1-Cmyc2-His6 (SEQ ID NO: 15), asdetermined using, eg., surface plasmon resonance.

An antibody of the invention may be capable of specifically bindingTREM-1, wherein said antibody is capable of specifically binding (i) atleast one amino acid residue selected from the group consisting of theA21, T22, K23, L24, T25, E26, and (ii) at least one amino acid residueselected from the group consisting of the A49, S50, S51, Q52, K53, A54,W55, Q56, I57, I58, R59, D60, G61, E62, M63, P64, K65, T66, L67, A68,C69, T70, E71, R72, P73, S74, K75, N76, S77, H78, P79, V80, Q81, V82,G83, R84, I85 and (iii) at least one amino acid residue selected fromthe group consisting of the C113, V114, I115, Y116, Q117, P118 and P119of human TREM-1.

An antibody of the invention may be capable of specifically binding apolypeptide comprising amino acids D38 to F48 of SEQ ID NO: 1 (humanTREM-1), as determined using, eg., HX-MS.

An antibody of the invention may have an epitope comprising one, two,three, four, five, six, seven or all of the amino acid residues D38,V39, K40, C41, D42, Y43, T44 and L45 of SEQ ID NO: 1 (human TREM-1) andone, two or all of the amino acid residues selected from the groupconsisting of the E46, K47 and F48 of SEQ ID NO: 1 (human TREM-1) asdetermined using, eg., HX-MS.

An antibody of the invention may have an epitope comprising one, two,three or all of the amino acid residues selected from the groupconsisting of the D42, E46, D92 and H93 of SEQ ID NO: 1 (human TREM-1),as determined using variants of TREM-1 and surface plasmon resonance.

An antibody of the invention may have an epitope comprising at least theamino acid residues E46 and/or D92 of SEQ ID NO: 1 (human TREM-1), asdetermined using variants of TREM-1 and surface plasmon resonance.

An antibody of the invention may further comprise one, two or all of theamino acid residues selected from the group consisting of the L31, I86and V101 of SEQ ID NO: 1 (human TREM-1).

An antibody of the invention may be capable of specifically binding apolypeptide comprising amino acid residues E19 to L26 of cynomolgusmonkey TREM-1 (SEQ ID NO: 12), or the corresponding amino acids of SEQID NO: 21, as determined using, eg., HX-MS.

An antibody of the invention may be capable of specifically bindinghuman TREM-1, wherein the epitope of said antibody comprises one, two,three, four, five, six, seven, eight, nine or all of the amino acidresidues selected from the group consisting of the V39, K40, C41, D42,Y43, L45, E46, K47, F48 and A49 of SEQ ID NO: 1. An antibody of theinvention may be capable of specifically binding human TREM-1, whereinthe epitope of said antibody comprises one, two, three, four, five, six,seven, eight, nine or all of the amino acid residues K40, D42, T44, L45,E46, K47, Y90, H91, D92, H93, G94, L95 and R97 of SEQ ID NO: 1.

An antibody of the invention may be capable of specifically bindinghuman TREM-1, wherein the epitope of said antibody comprises the D42 ofSEQ ID NO: 1. An antibody of the invention may be capable ofspecifically binding human TREM-1, wherein the epitope of said antibodycomprises the E46 of SEQ ID NO: 1. The epitope of said antibody maycomprise the V39, C41, D42, Y43, L45 of SEQ ID NO: 1. The epitope ofsaid antibody may comprise the E46, K47 and A49 of SEQ ID NO: 1. Theepitope of said antibody may further comprise the F48 of SEQ ID NO: 1.The epitope of said antibody may comprise the K40, 142T44, L45, E46,K47, Y90, H91, D92, H93, G94, L95 and R97 of SEQ ID NO: 1.

The definition of the term “paratope” is derived from the abovedefinition of “epitope” by reversing the perspective. Thus, the term“paratope” refers to the area or region on the Ab to which an Agspecifically binds, i.e. with which it makes physical contact to the Ag.

In the context of an X-ray derived crystal structure, defined by spatialcoordinates of a complex between an Ab, such as a Fab fragment, and itsAg, the term paratope is herein, unless otherwise specified orcontradicted by context, specifically defined as Ag residuescharacterized by having a heavy atom (i.e. a non-hydrogen atom) within adistance of 4 A from a heavy atom in TREM-1.

The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair maybe identified by routine methods. For example, the general location ofan epitope may be determined by assessing the ability of an antibody tobind to different fragments or variant TREM-1 polypeptides. The specificamino acids within TREM-1 that make contact with an antibody (epitope)and the specific amino acids in an antibody that make contact withTREM-1 (paratope) may also be determined using routine methods. Forexample, the antibody and target molecule may be combined and the Ab:Agcomplex may be crystallised. The crystal structure of the complex may bedetermined and used to identify specific sites of interaction betweenthe antibody and its target.

Antibodies that bind to the same antigen can be characterised withrespect to their ability to bind to their common antigen simultaneouslyand may be subjected to “competition binding”/“binning”. In the presentcontext, the term “binning” refers to a method of grouping antibodiesthat bind to the same antigen. “Binning” of antibodies may be based oncompetition binding of two antibodies to their common antigen in assaysbased on standard techniques such as surface plasmon resonance (SPR),ELISA or flow cytometry.

An antibody's “bin” is defined using a reference antibody. If a secondantibody is unable to bind to an antigen at the same time as thereference antibody, the second antibody is said to belong to the same“bin” as the reference antibody. In this case, the reference and thesecond antibody competitively bind the same part of an antigen and arecoined “competing antibodies”. If a second antibody is capable ofbinding to an antigen at the same time as the reference antibody, thesecond antibody is said to belong to a separate “bin”. In this case, thereference and the second antibody do not competitively bind the samepart of an antigen and are coined “non-competing antibodies”.

Antibody “binning” does not provide direct information about theepitope. Competing antibodies, i.e. antibodies belonging to the same“bin” may have identical epitopes, overlapping epitopes or even separateepitopes. The latter is the case if the reference antibody bound to itsepitope on the antigen takes up the space required for the secondantibody to contact its epitope on the antigen (“steric hindrance”).Non-competing antibodies generally have separate epitopes.

An antibody of the invention may compete with mAb 0170 for binding tohuman TREM-1. An antibody of the invention may compete with mAb 0170 forbinding to cynomolgus monkey TREM-1. In other words, an antibody of theinvention may belong to the same “bin” as mAb 0170.

The term “binding affinity” herein refers to a measurement of thestrength of a non-covalent interaction between two molecules, e.g. anantibody, or fragment thereof, and an antigen. The term “bindingaffinity” is used to describe monovalent interactions (intrinsicactivity).

Binding affinity between two molecules, e.g. an antibody, or fragmentthereof, and an antigen, through a monovalent interaction may bequantified by determination of the equilibrium dissociation constant(K_(D)). In turn, K_(D) can be determined by measurement of the kineticsof complex formation and dissociation, e.g. by the SPR method. The rateconstants corresponding to the association and the dissociation of amonovalent complex are referred to as the association rate constantk_(a) (or k_(on)) and dissociation rate constant k_(d) (or k_(off)),respectively. K_(D) is related to k_(a) and k_(d) through the equationK_(D)=k_(d)/k_(a).

Following the above definition, binding affinities associated withdifferent molecular interactions, such as comparison of the bindingaffinity of different antibodies for a given antigen, may be compared bycomparison of the K_(D) values for the individual antibody/antigencomplexes.

An antibody of the invention may bind human TREM-1 with an affinity (KD)that is 1×10⁻⁷M or less, 1×10⁻⁸ M or less, or 1×10⁻⁹ M or less, or1×10⁻¹⁰ M or less, 1×10⁻¹¹ M or less, 1×10⁻¹² M or lessor 1×10⁻¹³ M orless, as determined using surface plasmon resonance. An antibody of theinvention may bind cynomolgus monkey TREM-1 with an affinity (KD) thatis 1×10⁻⁷ M or less, 1×10⁻⁸M or less, or 1×10⁻⁹ M or less, or 1×10⁻¹⁰ Mor less, 1×10⁻¹¹ M or less, 1×10⁻¹² M or less or 1×10⁻¹³ M or less, asdetermined using surface plasmon resonance.

The term “binding specificity” herein refers to the interaction of amolecule such as an antibody, or fragment thereof, with a singleexclusive antigen, or with a limited number of highly homologousantigens (or epitopes). In contrast, antibodies that are capable ofspecifically binding to TREM-1 are not capable of binding dissimilarmolecules. Antibodies according to the invention may not be capable ofbinding Nkp44.

The specificity of an interaction and the value of an equilibriumbinding constant can be determined directly by well-known methods.Standard assays to evaluate the ability of ligands (such as antibodies)to bind their targets are known in the art and include, for example,ELISAs, Western blots, RIAs, and flow cytometry analysis. The bindingkinetics and binding affinity of the antibody also can be assessed bystandard assays known in the art, such as SPR.

A competitive binding assay can be conducted in which the binding of theantibody to the target is compared to the binding of the target byanother ligand of that target, such as another antibody.

In another aspect, the present invention provides compositions andformulations comprising molecules of the invention, such as the TREM-1antibodies, polynucleotides, vectors and cells described herein. Forexample, the invention provides a pharmaceutical composition thatcomprises one or more TREM-1 antibodies of the invention, formulatedtogether with a pharmaceutically acceptable carrier.

Accordingly, one object of the invention is to provide a pharmaceuticalformulation comprising such a TREM-1 antibody which is present in aconcentration from 0.25 mg/ml to 250 mg/ml, such as a concentration offrom 10 to 200 mg/ml, and wherein said formulation has a pH from 2.0 to10.0, such as a pH of from 40 to 8.0. The formulation may furthercomprise one or more of a buffer system, a preservative, a tonicityagent, a chelating agent, a stabilizer and/or a surfactant, as well asvarious combinations thereof. The use of preservatives, isotonic agents,chelating agents, stabilizers and surfactants in pharmaceuticalcompositions is well-known to the skilled person. Reference may be madeto Remington: The Science and Practice of Pharmacy, 19^(th) edition,1995.

In one embodiment, the pharmaceutical formulation is an aqueousformulation. Such a formulation is typically a solution or a suspension,but may also include colloids, dispersions, emulsions, and multi-phasematerials. The term “aqueous formulation” is defined as a formulationcomprising at least 50% w/w water. Likewise, the term “aqueous solution”is defined as a solution comprising at least 50% w/w water, and the term“aqueous suspension” is defined as a suspension comprising at least 50%w/w water.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, to which the physician or the patient adds solvents and/ordiluents prior to use.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of such an antibody, and a buffer, wherein the antibody ispresent in a concentration from 1 mg/ml or above, and wherein saidformulation has a pH from about 2.0 to about 10.0.

The TREM-1 antibodies of the present invention and pharmaceuticalcompositions comprising such antibodies may be used for the treatment ofinflammatory diseases such as the following: inflammatory bowel disease(IBD), Crohns disease (CD), ulcerative colitis (UC), irritable bowelsyndrome, rheumatoid arthritis (RA), juvenile idiopathic arthritis(JIA), psoriasis, psoriatic arthritis, systemic lupus erythematosus(SLE), lupus nephritis, type I diabetes, Grave's disease, multiplesclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronaryartery disease, chronic obstructive pulmonary disease, interstitial lungdisease, autoimmune thyroiditis, scleroderma, systemic sclerosis,osteoarthritis, atoptic dermatitis, vitiligo, graft versus host disease,Sjogrens's syndrome, autoimmune nephritis, Goodpasture's syndrome,chronic inflammatory demyelinating polyneuropathy, allergy, asthma andother autoimmune diseases that are a result of either acute or chronicinflammation.

TREM-1 antibodies of the invention may be suitable for use in thetreatment of individuals with inflammatory bowel disease. InflammatoryBowel Disease (IBD) is a disease that may affect any part of thegastrointestinal tract from mouth to anus, causing a wide variety ofsymptoms. IBD primarily causes abdominal pain, diarrhea (which may bebloody), vomiting or weight loss, but may also cause complicationsoutside of the gastrointestinal tract such as skin rashes, arthritis,inflammation of the eye, fatigue and lack of concentration. Patientswith IBD can be divided into two major classes, those with ulcerativecolitis (UC) and those with Crohn's disease (CD). CD generally involvesthe ileum and colon, it can affect any region of the intestine but isoften discontinuous (focused areas of disease spread throughout theintestine). UC always involves the rectum (colonic) and is morecontinuous. In CD, the inflammation is transmural, resulting inabscesses, fistulas and strictures, whereas in UC, the inflammation istypically confined to the mucosa. There is no known pharmaceutical orsurgical cure for Crohn's disease, whereas some patients with UC can becured by surgical removal of the colon. Treatment options are restrictedto controlling symptoms, maintaining remission and preventing relapse.Efficacy in inflammatory bowel disease in the clinic may be measured asa reduction in the Crohn's Disease Activity Index (CDAI) score for CDwhich is scoring scale based on laboratory tests and a quality of lifequestionnaire. In animal models, efficacy is mostly measured by increasein weight and also a disease activity index (DAI), which is acombination of stool consistency, weight and blood in stool.

TREM-1 antibodies of the invention may be suitable for use in thetreatment of individuals with rheumatoid arthritis. Rheumatoid arthritis(RA) is a systemic disease that affects nearly if not all of the bodyand is one of the most common forms of arthritis. It is characterized byinflammation of the joint, which causes pain, stiffness, warmth, rednessand swelling. This inflammation is a consequence of inflammatory cellsinvading the joints, and these inflammatory cells release enzymes thatmay digest bone and cartilage. As a result, this inflammation can leadto severe bone and cartilage damage and to joint deterioration andsevere pain, among other physiologic effects. The involved joint canlose its shape and alignment, resulting in pain and loss of movement.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop an inflammatory arthritis that resembles human rheumatoidarthritis. Since CIA shares similar immunological and pathologicalfeatures with RA, this makes it a suitable model for screening potentialhuman anti-inflammatory compounds. Efficacy in this model is measured bydecrease in joint swelling. Efficacy in RA in the clinic is measured bythe ability to reduce symptoms in patients which is measured as acombination of joint swelling, eythrocyte sedimentation rate, C-reactiveprotein levels and levels of serum factors, such as anti-citrullinatedprotein antibodies.

TREM-1 antibodies of the invention may be suitable for use in thetreatment of individuals with psoriasis. Psoriasis is a T-cell mediatedinflammatory disorder of the skin that can cause considerablediscomfort. It is a disease for which there is currently no cure and itaffects people of all ages. Although individuals with mild psoriasis canoften control their disease with topical agents, more than one millionpatients worldwide require ultraviolet light treatments or systemicimmunosuppressive therapy. Unfortunately, the inconvenience and risks ofultraviolet radiation and the toxicities of many therapies limit theirlong-term use. Moreover, patients usually have recurrence of psoriasis,and in some cases rebound shortly after stopping immunosuppressivetherapy. A recently developed model of psoriasis based on the transferof CD4+ T cells mimics many aspects of human psoriasis and therefore canbe used to identify compounds suitable for use in treatment of psoriasis(Davenport et al., Internat. Immunopharmacol 2:653-672, 2002). Efficacyin this model is a measured by reduction in skin pathology using ascoring system. Similarly, efficacy in patients is measured by adecrease in skin pathology.

TREM-1 antibodies of the invention may be suitable for use in thetreatment of individuals with psoriatic arthritis. Psoriatic arthritis(PA) is a type of inflammatory arthritis that occurs in a subset ofpatients with psoriasis. In these patients, the skin pathology/symptomsare accompanied by a joint swelling similar to that seen in rheumatoidarthritis. It features patchy, raised, red areas of skin inflammationwith scaling. Psoriasis often affects the tips of the elbows and knees,the scalp, the navel and around the genital areas or anus. Approximately10% of patients who have psoriasis also develop an associatedinflammation of their joints.

The term “treatment”, as used herein, refers to the medical therapy ofany human or other animal subject in need thereof. Said subject isexpected to have undergone physical examination by a medical orveterinary medical practitioner, who has given a tentative or definitivediagnosis which would indicate that the use of said treatment isbeneficial to the health of said human or other animal subject. Thetiming and purpose of said treatment may vary from one individual toanother, according to many factors, such as the status quo of thesubject's health. Thus, said treatment may be prophylactic, palliative,symptomatic and/or curative.

In terms of the present invention, prophylactic, palliative, symptomaticand/or curative treatments may represent separate aspects of theinvention.

An antibody of the invention may be administered parenterally, such asintravenously, such as intramuscularly, such as subcutaneously.Alternatively, an antibody of the invention may be administered via anon-parenteral route, such as perorally or topically. An antibody of theinvention may be administered prophylactically. An antibody of theinvention may be administered therapeutically (on demand).

EXEMPLARY EMBODIMENTS

1. A method of identifying a functional TREM-1 antibody, comprising (a)culturing a first cell expressing TREM-1, a signalling protein and areporter construct; (b) measuring the activity of the first cell whensaid cell is incubated with a TREM-1 modifying agent; (c) contacting theculture of (b) with a TREM-1 antibody; and (d) measuring that theactivity of the first cell is less than or more than the activitymeasured in (b).

2. A method of identifying a blocking TREM-1 antibody, comprising (a)culturing a first cell expressing TREM-1, a signalling protein and areporter construct; (b) measuring the activity of the first cell whensaid cell is incubated with an activated neutrophil; (c) contacting theculture of the first cell and the activated neutrophil with a TREM-1antibody; and (d) measuring that the activity of the first cell is lessthan the activity measured in (b).

3. The method of any one of embodiments 1-2, wherein the modifying agentof (b) is an activated neutrophil or a TREM-1 ligand.

4. A method of identifying a stimulating TREM-1 antibody, comprising (a)culturing a first cell expressing TREM-1, a signalling protein and areporter construct; (b) measuring the activity of the first cell; (c)contacting/incubating said cell with a TREM-1 antibody, and (d)measuring that the activity of the first cell is more than the activitymeasured in (b).

5. The method of any one of embodiments 1-4, wherein the first cell isof haematopoetic origin.

6. The method according to embodiment 5, wherein the cell ofhaematopoetic origin is a myeloid cell.

7. The method according to embodiment 5, wherein the cell ofhaematopoetic origin is a T-cell.

8. The method according to any one of embodiments 1-7, wherein thesignalling protein is DAP10.

9. The method according to any one of embodiments 1-7, wherein thesignalling protein is DAP12.

10. The method according to any one of embodiments 1-7, wherein thesignalling protein is TCR zeta.

11. The method according to any one of embodiments 1-7, wherein thesignalling protein is Fc gamma RIII.

12. The method according to any one of embodiments 1-7, wherein thesignalling protein is a Fc receptor.

13. The method according to any one of embodiments 1-6, wherein thereporter construct comprises a transcription factor and a reporter gene.

14. The method according to embodiment 13, wherein said transcriptionfactor is NFAT.

15. The method according to embodiment 14, wherein said transcriptionfactor is NFkB.

16. The method according to any one of embodiments 13-15, wherein saidreporter gene encodes β-galactosidase.

17. The method according to any one of embodiments 13-15, wherein saidreporter gene encodes luciferase.

18. The method according to any one of embodiments 13-15, wherein saidreporter gene encodes green fluorescent protein (GFP).

19. The method according to any one of embodiments 13-15, wherein saidreporter gene is a gene that encodes chloramphenicol transferase.

20. A method of identifying a blocking TREM-1 antibody, comprising (a)culturing a T-cell expressing TREM-1, DAP12 and a gene that encodesluciferase; (b) measuring the luminescence of the T-cell when it isincubated with an activated neutrophil; (c) contacting the co-culture of(b) with a TREM-1 antibody; and (d) measuring that the luminescence ofthe T-cell is less than the activity measured in (b).

21. The method according to embodiment 7, wherein said cell is aBWZ.36/hTREM-1:DAP12:NFAT-LacZ T-cell line.

22. The antibody identified by the method of any one of embodiments 1-3and 5-21.

23. An antibody that is capable of specifically binding to TREM-1 andthat is capable of blocking TREM-1 function.

24. The antibody according to any one of embodiments 22-23, wherein saidantibody is capable of preventing or reducing thedimerisation/multimerisation of TREM-1.

25. The antibody according to any one of embodiments 22-24, wherein saidantibody is capable of blocking the interaction between TREM-1 and itsligand.

26. The antibody according to any one of embodiments 22-25, wherein saidantibody is capable of blocking PGLYRP1-induced TREM-1 function.

27. The antibody according to any one of embodiments 22-26, wherein theTREM-1 is human TREM-1.

28. The antibody according to embodiment 27, wherein said antibody isalso capable of specifically binding to and blocking the function ofTREM-1 from another species than a human.

29. The antibody according to embodiment 28, wherein the TREM-1 fromanother species is cynomolgus monkey TREM-1.

30. The antibody according to embodiment 28, wherein the TREM-1 fromanother species is rhesus monkey TREM-1.

31. The antibody according to any one of embodiments 22-30, which iscapable of specifically binding K20A-hTREM-1-Cmyc2-His6 (SEQ ID NO: 13).

32. The antibody according to any one of embodiments 22-31, which iscapable of specifically bindingA24T/Y28F/N30S/R32Q/P70H-cTREM-1-Cmyc2-His6 (SEQ ID NO: 14).

33. The antibody according to any one of embodiments 22-32, which iscapable of specifically bindingA24T/Y28F/N30S/R320/E54K-cTREM-1-Cmyc2-His6 (SEQ ID NO: 15).

34. The antibody according to any one of embodiments 22-33, whichcompetes with mAb 0170 for binding to human TREM-1.

35. The antibody according to any one of embodiments 22-34, whichcompetes with mAb 0170 for binding to cynomolgus monkey TREM-1.

36. The antibody according to any one of embodiments 22-35, which iscapable of specifically binding a polypeptide comprising amino acids D38to F48 of SEQ ID NO: 1 (human TREM-1), as determined using, eg., HX-MS.

37. The antibody according to any one of embodiments 22-36, which has anepitope comprising one, two, three, four, five, six, seven or all of theamino acid residues selected from the group consisting of the D38, V39,K40, C41, D42, Y43, T44 and L45 of SEQ ID NO: 1 (human TREM-1) and one,two or all of the amino acid residues selected from the group consistingof the E46, K47 and F48 of SEQ ID NO: 1 (human TREM-1), as determinedusing, eg., HX-MS.

38. The antibody according to any one of embodiments 22-37, which iscapable of specifically binding a polypeptide comprising amino acidresidues E38 to L45 of cynomolgus monkey TREM-1, as determined using,eg., HX-MS.

39. The antibody according to any one of embodiments 22-38 which has anepitope comprising at least the amino acid residues selected from thegroup consisting of the D42, E46, D92 and H93 of SEQ ID NO: 1 (humanTREM-1), as determined using surface plasmon resonance.

40. The antibody according to any one of embodiments 22-39 which has anepitope comprising at least the amino acid residues E46 and/or D92 ofSEQ ID NO: 1 (human TREM-1), as determined using surface plasmonresonance.

41. The antibody according to any one of embodiments 22-40, wherein saidantibody is capable of specifically binding (i) at least one amino acidresidue selected from the group consisting of the A21, T22, K23, L24,T25, E26, and (ii) at least one amino acid residue selected from thegroup consisting of the A49, S50, S51, Q52, K53, A54, W55, Q56, I57,I58, R59, D60, G61, E62, M63, P64, K65, T66, L67, A68, C69, T70, E71,R72, P73, S74, K75, N76, S77, H78, P79, V80, Q81, V82, G83, R84, I85 and(iii) at least one amino acid residue selected from the group consistingof the C113, V114, I115, Y116, Q117, P118 and P119 of human TREM-1.

42. The antibody according to any one of embodiments 22-40, wherein saidantibody is capable of specifically binding (i) at least one amino acidresidue selected from the group consisting of the V39, K40, C41, D42,Y43, T44, L45, E46, K47, F48, A49, S50, S51, Q52, K53, A54, W55, Q56,and (ii) at least one amino acid residue selected from the groupconsisting of the T70, E71, R72, P73, S74, K75, N76, S77, H78, P79, V80,Q81, V82, G83, R84, I85 and (iii) at least one amino acid residueselected from the group consisting of the and C113, V114, I115, Y116,Q117, P118, P119.

43. The antibody according to any one of embodiments 22-35, wherein saidantibody is capable of specifically binding human TREM-1, wherein theepitope of said antibody comprises one, two, three, four, five, six,seven, eight, nine or all of the amino acid residues K40, D42, T44, L45,E46, K47, Y90, I191, D92, H93, G94, L95 and R97 of SEQ ID NO: 1.

44. The antibody according to any one of embodiments 22-35, wherein saidantibody is capable of specifically binding human TREM-1, wherein theepitope of said antibody comprises the K40, D42, T44, L45, E46, K47,Y90, H91, D92, H93, G94, L95 and R97 of SEQ ID NO: 1.

45. The antibody according to any one of embodiments 22-44, the heavychain of which comprises a CDRH3 sequence corresponding to amino acidresidues 101 to 110 (DMGIRRQFAY) of SEQ ID NO: 4, wherein one, two orthree of said amino acid residues may be substituted by a differentamino acid.

46. The antibody according to any one of embodiments 22-44, the heavychain of which comprises a CDRH3 sequence corresponding to amino acidresidues 101 to 110 (DMGQRRQFAY) of SEQ ID NO: 6, wherein one, two orthree amino acid residues may be substituted by a different amino acid.

47. The antibody according to any one of embodiments 45-46, furthercomprising a CDRH1 sequence corresponding to amino acid residues 31 to35 (TYAMH) of SEQ ID NO: 4 or SEQ ID NO: 6, wherein one of these aminoacid residues may be substituted by a different amino acid residue;and/or a CDRH2 sequence corresponding to amino acids 50 to 68(RIRTKSSNYATYYAASVKG) of SEQ ID NO: 4 or SEQ ID NO: 6, wherein one, twoor three of said amino acids may be substituted by a different aminoacid residue.

48. The antibody according to any one of embodiments 45-47, the lightchain of which comprises: a CDRL1 sequence corresponding to amino acidresidues 24 to 38 (RASESVDTFDYSFLH) of SEQ ID NO: 5 or SEQ ID NO: 7,wherein one, two or three of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL2 sequencecorresponding to amino acid residues 54 to 60 (RASNLES) of SEQ ID NO: 5or SEQ ID NO: 7, wherein one or two of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL3 sequencecorresponding to amino acid residues 93 to 101 (QQSNEDPYT) of SEQ ID NO:5 or SEQ ID NO: 7, wherein one or two of these amino acid residues maybe substituted with a different amino acid.

49. The antibody according to any one of embodiments 45-48, comprisingSEQ ID NO. 4 or SEQ ID NO: 6 and/or SEQ ID NO: 5 or SEQ ID NO: 7.

50. The antibody according to any one of embodiments 22-49, which bindshuman TREM-1 with a binding affinity (KD) that is 1×10⁻⁷ M or less,1×10⁻⁸ M or less, or 1×10⁻⁹ M or less, or 1×10⁻¹⁰ M or less, 1×10⁻¹¹ Mor less, 1×10⁻¹² M or less or 1×10⁻¹³ M or less, as determined usingsurface plasmon resonance.

51. The antibody according to embodiment 50, wherein said bindingaffinity (KD) is 1×10⁻¹⁰ M or less.

52. The antibody according to any one of embodiments 22-51, which bindscynomolgus monkey TREM-1 with a binding affinity (KD) that is 1×10⁻⁷ Mor less, 1×10⁻⁸ M or less, or 1×10⁻⁹M or less, or 1×10⁻¹⁰ M or less,1×10⁻¹¹ M or less, 1×10⁻¹²M or less or 1×10³M or less, as determinedusing surface plasmon resonance.

53. The antibody according to embodiments 52, wherein said bindingaffinity is 1×10⁻⁹ M or less.

54. The antibody according to any one of embodiments 22-53, which is anIgG.

55. The antibody according to any one of embodiments 22-54 for use as amedicament.

56. The antibody according to any one of embodiments 22-55 for thetreatment of an autoimmune disease and/or chronic inflammation.

57. The antibody according to any one of embodiments 22-54 for themanufacture of a medicament for the treatment of an autoimmune diseaseand/or a chronic inflammation.

58. A method of treating an autoimmune disease and/or chronicinflammation comprising administering an antibody according to any oneof embodiments 22-54 to a subject in need thereof.

59. The use according to any one of embodiments 55-57 or the methodaccording to embodiment 58, wherein said autoimmune disease isrheumatoid arthritis.

60. The use according to any one of embodiments 55-57 or the methodaccording to embodiment 58, wherein said autoimmune disease is Crohn'sdisease.

61. The use according to any one of embodiments 55-57 or the methodaccording to embodiment 58, wherein said autoimmune disease isulcerative colitis.

62. The use according to any one of embodiments 55-57 or the methodaccording to embodiment 58, wherein said autoimmune disease is psoriaticarthritis.

63. The use according to any one of embodiments 55-57 or the methodaccording to embodiment 58, wherein said autoimmune disease is juvenileidiopathic arthritis.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1: Generation of a BWZ.36 humanTREM-1:DAP12 Stable CellLine

The BWZ.36/hTREM-1:DAP12:NFAT-LacZ cell line (herein also referred to asthe “BWZ/hTREM-1 reporter cell”) was derived from BW5147 T cells (Musmusculus thymus lymphoma cell line. ATCC TIB-47, LGC Standards,Middelsex, UK) and contains a LacZ reporter construct regulated by fourcopies of the NFAT promoter element (see Karttunen, J. & Shastri, N.(1991) Proc. Natl. Acad. Sci. USA 88, 3972-3976 and Fiering, S.,Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. &Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834). TREM/DAP12/pMX-IRESvector (encoding 786 bp of TREM-1 from a SmaI site to BamHI site usingTREM-1 cDNA (Gene Bank Ref. ID: NM_018643.2, Sino Biological Inc.,Beijing, China) as template and oligo 5′ TAGTAGGGATCCGCTGGTGCACAGGAAGG(SEQ ID NO: 16) and 5′ TAGTAGGCGGCCGCTTCGTGGCCTAGGGAC (SEQ ID NO: 17) asprimers cloned into pREShyg vector GenBank Accession #U89672 (Cat. no.6061-1, Clontech Laboratories, CA, USA) was transfected in PLAT-Epackaging cell line (provided by W. Yokoyama, Washington University;alternatively, Cat. no. RV-101, Cell Biolabs Inc, Bio-Mediator KY,Vantaa, Finland) using Superfect transfection reagent (Cat. no. 301305,Qiagen Nordic, Denmark). PLAT-E supernatants containingTREM/DAP12/pMX-IRES viral particles were used to infect BWZ.36 cells asfollows: 2×10⁵ BWZ.36 cells were cultured in 6 well plates and themedium was replaced with 1.5 ml of supernatant containing the viralparticles+8 mg/ml of polybrene. After 6-8 hours, 1.5 ml of normal mediumwas added to the plate and the cells were incubated for an additional 24hours. BWZ.36 cell lines stably expressing TREM-1 were stained with antiTREM-1 monoclonal antibody (clone 21C7) and isolated by cell sorting.

Example 2: Cultivation of a BWZ.36 humanTREM-1:DAP12 Stable Cell Line

BWZ/hTREM-1 reporter cell were cultured in RPMI 1640 w/o phenol red (Cat#11835, Gibco, Carlsbad Calif., USA), supplemented with 10% FCS (Cat#16140-071, Gibco, New York, USA), 1% Pen/Strep (Cat #15070-06, Gibco),1 mM Sodium Pyruvate (Cat #11360, Gibco), 5 μM-2ME (Cat #31350-010,Gibco) and 2 mM L-Glutamine (Cat #25030, Gibco). No special plates orcoating was required. 10 ml Versene (Cat #15040, Gibco) was added todetach the cells which then were transferred to tubes, centrifuged 1200rpm 5 min and washed in fresh RPMI 1640 w/o phenol red. These cell werethen ready to use in an assay or re-culture for further propagation.

Example 3: Immunisation of Mice and Identification of mAbs

In order to generate antibodies that bind human TREM-1, both wild typeBalb/C mice and TREM-1 knock-out (KO) mice (C57BL/6 background) wereimmunised with either human (h) TREM-1 (SEQ ID NO:1), cells expressinghTREM-1 (BWZ.36 cells), or a combination of both. Primary screening wasdone either by means of direct ELISA on hTREM-1 protein or by means ofFMAT, using BWZ.36 cells expressing hTREM-1. Secondary screening wasdone by flow cytometry on HEK293 cells expressing hTREM-1. Positivehybridoma supernatants were then screened in the BWZ/hTREM-1 reporterassay described in Example 4.

The highest number of blocking antibodies was obtained from KO miceimmunised with hTREM-1 protein six times at two weeks intervals,followed by a booster injection. In total, over 200 hTREM-1 antibodieswere isolated, of which approximately 70 were subsequently found to havea blocking effect.

All TREM-1 specific hybridoma supernatants were tested in theBWZ/hTREM-1 reporter assay first as supernatants and later as purifiedantibodies, in full titration from 5000 ng/ml down to 7 ng/ml, both assoluble and as platebound antibodies. Blood from a range of differentdonors was used as a source of fresh neutrophils. As an example, FIG. 4shows antibodies from one fusion where the activity in the reporterassay as blocking activity is on the x-axis and the agonistic activitywhen the antibody is plate-bound is on the y-axis.

Example 4: Identification of PGLYRP1 as a Neutrophil-Expressed TREM-1Ligand

PGLYRP1 was identified as a TREM-1 ligand through the use ofimmunoprecipitation coupled with mass spectroscopy (IP-MS). SolubleTREM-1 tetramer was used as an affinity “bait” molecule to identify aligand. Briefly, TREM-1-tetramer-Fc (SEQ ID NO: 2) and separatelyCD83-Fc (SEQ ID NO: 5) were each incubated at final concentrations of100 μg/ml with 270 million human neutrophils, purified by dextransedimentation as described above, in 1 mL PBS at 4° C., 90 minutes withmild shaking. After pelleting these cells, the cells were resuspended in1 mL PBS buffer with the inclusion of the crosslinker3,3′-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) (Thermo Scientific:21578, Rockford, Ill., USA), at a concentration of 2 mM and incubated 30minutes at room temperature, Cells were washed 3× with 1 mL PBS followedby lysis in 1 mL RIPA buffer (Thermo Scientific, 89901, Rockford, Ill.,USA). The lysate was centrifuged at 15,000×g for 10 minutes at 4° C. toremove insoluble materials. Neutrophil proteins cross-linked to Fccoupled probes were immunoprecipitated from the supernatant usingProtein A Mag Sepharose™ beads (GE Healthcare Life Sciences, 28-9670-56,Piscataway, N.J., USA). Briefly, 50 μL of beads were first washed with200 μL PBS, then resuspended in 1 mL of cell lysate, incubated 60minutes at 4° C., magnetically captured, and sequentially washed 2× with200 μl RIPA buffer then 3× with 200 μL PBS. Upon removing PBS from thefinal magnetic capture, proteins were eluted from the magnetic beadsusing 200 μL buffer containing 8 M Urea, 100 mM Tris (pH 8.0), and 15 mMTCEP (Thermo Scientific, 77720, Rockford, Ill., USA) and incubated atroom temperature for 30 minutes, beads were captured and supernatant wastransferred to a Microcon Ultracel YM-30 filter (Millipore, 42410,Billerica, Mass., USA). Samples were spun at 14, 000×g, 20° C., 30-60minutes until no liquid remained on the top of the filter membrane. Theretained proteins were then alkylated with 100 μL 50 mM IAA(iodoacetamide) in 8 M Urea for 30 minutes in dark at room temperature.The filter was washed 2× with 100 μL 50 mM NH₄HCO₃ and then transferredto a new collection tube. 1 μg trypsin (Promega, V5111, Madison, Wis.)in 60 μL 50 mM NH₄HCO₃ was added followed by incubation at 37° C.overnight. The tryptic digest was collected by centrifugation at14,000×g for 30 minutes followed by washing the filter with 50 μL 50 mMNH₄HCO₃. 10 μL of the digest was analyzed by LC/MS/MS using anLTQ-Orbitrap-XL mass spectrometer (Thermo Scientific, Waltham, Mass.,USA). The data was searched against 1PI human database (v3.81) usingSEQUEST-Sorcerer engine (4.0.4 build) (SageN, Milpitas, Calif., USA) andthen post processed with Scaffold 3 (Proteome Software, Portland, Oreg.,USA) to filter protein IDs with a false discovery rate of 1%. Afternegative control subtraction, PGLYRP1 was found to be a high-confidenceprotein specifically associated with hTREM-1 tetramer. Theimmunoprecipitation in the neutrophils showed that out of the 148identified proteins, 72 proteins were immunoprecipitated by the controlconstruct (CD83) alone, 73 of the proteins were identical for TREM-1 andCD83, whereas only three were TREM-1 specific (FIG. 3). The experimentwas subsequently repeated using neutrophils from a different donor andPGLYRP1 was again identified as specifically interacting with hTREM-1.

Example 5: Refolding and Purification of Human PGLYRP1 Expressed from E.coli

Human PGLYRP1 was expressed as inclusion bodies in Escherichia coli BL21(DE3) cells. Bacteria were harvested by centrifugation, resuspended in50 mM Tris-HCl pH8.0, 500 mM NaCl, 5 mM EDTA, 0.5% Triton X-100 anddisrupted by sonication. The insoluble pellet was washed three timeswith 50 mM Tris, pH 8.0, 1% TritonX-100, 2 M urea and once with 50 mMTris pH 8.0, then solubilized in 50 mM Tris-HCl, 6M guanidinehydrochloride, pH7.4, 1 mM DTT (final protein concentration 20 mg/ml).For in vitro folding, solubilized human PGLYRP1 inclusion bodies werediluted into 50 mM Tris, pH 8.0, 2 mM EDTA, 5 mM cysteamine, 0.5 mMcystamine, 0.4 M arginine (final protein concentration 1 mg/ml). Afterovernight at 4° C., the folding mixture was cleared bycentrifugation/filtration and then diluted 12 fold into 10 mM MES pH 3.5to lower the conductivity and pH (final pH˜5.8, conductivity˜6 mS/cm).The diluted folding mixture was then applied to a Hitrap SP HP 5 mlcolumn (17-1151-01 GE Healthcare, Uppsala, Sweden), followed by a 5column volume wash with 50 mM MES pH 5.8. The bound human PGLYRP1 wasthen eluted with a 0-60% linear gradient of 50 mM MES pH 5.8, 1 M NaClin 20 column volume. The fractions containing refolded human PGLYRP1were pooled and concentrated to less than 4 ml by Amicon ultra 15centrifugal units ((UFC800324 3,000 kDa MWCO, Millipore, Hellerup,Denmark). A Hiload 26/60 Superdex 75 318 ml column ((17-1070-01 GEHealthcare, Uppsala, Sweden) was then used to polish and buffer-exchangethe proteins to Phosphate Buffered Saline (PBS). Majority of refoldedhuman PGLYRP1 proteins was in monomer form. After concentrating, thefinal protein concentration was determined by measuring 280 nmabsorbance with a NANODROP UV spectrometer. Protein purity was assessedby sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Example 6: Creation of a TREM-1 Responsive Reporter Assay

The TREM-1 reporter cell line was generated by transfecting the BWZ.36cell line with a NFAT-LacZ reporter construct, as well as hTREM-1 andDAP12 (as described in Example 1). Neutrophils of healthy donors werepurified by means of Dextran sedimentation. Blood was stratified onFicollPaque (17-0840-03, GE Healthcare, Piscataway, N.J., USA) gradientwith a rate of 3 parts of Ficoll and 4 parts of blood in a 50 ml tube,then centrifuged at 400×g for 30 minutes at 22° C., without brake. Theintermediate PBMC band was gently removed by aspiration. The neutrophilsstratified on the packed RBC were aspirated and transferred to a 50 mlpolypropylene tube. The neutrophils and contaminating RBCs were dilutedto 40 ml with 1×PBS and followed by addition of 10 ml 4% DEXTRAN 500(Sigma, 31392, St Louis, Mo., USA) in PBS solution. After mixing bygentle inversion, the tubes were left at 22° C. for 20-30 min. Agranulocyte rich supernatant was then transferred into a fresh tube andcentrifuged at 250×g. 5 min, 22° C.; the supernatant was aspirated anddiscarded. Contaminating RBCs were removed with an osmotic lysis,briefly, the cell pellet was resuspended in 7.5 mi of 0.2% NaCl; gentlymixed for 55-60 seconds and 17.5 ml of a 1.2% NaCl solution was added.The volume was then brought to 50 ml with PBS and spun at 250×g for 5min, the pellet was resuspended in 7.5 ml of 0.2% NaCl to repeat thelysis a second time. The final granulocyte pellet was resuspended inRPMI/10% FBS. These neutrophils were stimulated with PGN (InVivogen,tlrl-pgnsa, SanDiego, Calif., USA) overnight to generate activatedneutrophils able to stimulate TREM-1. The BWZ/hTREM-1 reporter cellswere then added to the PGN activated neutrophil cultures in a 1:3 ratioof reporter cell:neutrophils. Instead of activated neutrophils, a TREM-1ligand complex consisting of PGLYRP1 (SEQ ID NO: 23) and PGN could beused to stimulate TREM-1. The assay was run in Poly-D-Lysine coatedBlack cell culture plates (no. 356640 from BD Biosciences, San Jose,Calif., USA). TREM-1 activation was read out after 24 hours of cultureusing the BetaGlo reagent (E4720 from Promega, Madison, Wis., USA) andluminescence measured using a TopCount Luminescence counter from PerkinElmer. As positive control TREM-1 could be activated by a plate-boundTREM-1 antibody (R&D MAB1278, Minneapolis, Minn., USA) able to agoniseTREM-1. Plates were coated with isotype control or TREM-1 antibodyMAB1278 (3 ug/ml in PBS, 100 ul/well) in the fridge O/N or for 2 hr at37° C., 5% CO2 before the BWZ/hTREM-1 reporter cells were added. After6-24 hours incubation TREM-1 activation could be read using the BetaGloreagent (E4720 from Promega, Madison, Wis., USA) and luminescencemeasured using a TopCount Luminescence counter from Perkin Elmer. ThisBWZ.36/hTREM-1:DAP12:NFAT-LacZ cell line (the “BWZ/hTREM-1 reportercell”) showed to be highly responsive to antibody-mediated cross linkingof TREM-1, giving a ˜40-fold induction of the NFAT-driven LacZproduction when stimulated with 1-10 μg/ml plate bound commerciallyavailable anti-TREM-1 antibody, as compared to the isotype control (FIG.1). When stimulated with a toll-like receptor cocktail (tlrl-kit2hm,Invivogen, Sigma-Aldrich Denmark) alone (BWZ+TLR) no increase in signalwas observed. Furthermore, unactivated neutrophils could not stimulateTREM-1, whereas TLR agonist cocktail (tlrl-kit2hm, Invivogen,Sigma-Aldrich Denmark) activated neutrophils could stimulate theBW7/hTREM-1 reporter cell.

Table 1, below, shows that TREM-1 antibodies disclosed herein are ableto block the ligand-induced TREM-1 activation in such BWZ/hTREM-1reporter cell assay.

TABLE 1 Antibody Luminescence × 10E6 BWZ + activated neutrophils 8.8 MAB1278 10 HPA 7.9 TREM26 9.9 TREM37 6.1 14F128 0.3 14F69 0.4 14F116 0.714F11 0.4 14F113 0.3

None of the tested commercial available antibodies: MAB1278 (cat. no.MAB1278, R&D Systems, Minneapolis, Minn. 55413, USA), anti-TREM-1 HPA(cat. no. HPA005563, Sigma, St Louis, Mo., USA), TREM26 (cat. no.314902, Biolegend, San Diego, Calif. 92121, USA) and TREM37 (cat. no.316102, Biolegend, San Diego, Calif. 92121, USA) were able to block theTREM-1 signal.

Example 7: Epitope Mapping Using HX-MS Materials

Protein Batches Used were:

hTREM-1: human recombinant TREM-1, non-glycosylated, produced in E.coli. (cat. no. PRO-457, ProSpec-Tany TechnoGene Ltd., Rehovot, Israel).

TABLE 2 mAbs used Antibody Supplier mAb 0023 — mAb 0024 — mAb 0025 — mAb0026 — MAB1278 RnD Systems TREM26 BioLegend

All proteins were buffer exchanged to PBS pH 7.4 before experiments.

Methods: HX-MS Experiments Instrumentation and Data Recording

The HX experiments were automated by a Leap robot (H/D-x PAL: LeapTechnologies Inc.) operated by the LeapShell software (Leap TechnologiesInc.), which performed initiation of the deuterium exchange reaction,reaction time control, quench reaction, injection onto the UPLC systemand digestion time control. The Leap robot was equipped with twotemperature controlled stacks maintained at 20° C. for buffer storageand HX reactions and maintained at 2° C. for storage of protein andquench solution, respectively. The Leap robot furthermore contained acooled Trio VS unit (Leap Technologies Inc.) holding the pre- andanalytical columns as well as the pepsin column, the LC tubing andswitching valves at 1° C. The switching valves of the Trio VS unit havebeen upgraded from HPLC to Microbore UHPLC switch valves (Cheminert,VICI AG). For the inline pepsin digestion, 100 μL quenched samplecontaining 200 pmol hTREM-1 was loaded and passed over the Poroszyme®Immobilised Pepsin Cartridge (2.1×30 mm (Applied Biosystems)) using aisocratic flow rate of 200 μL/min (0.1% formic acid:CH₃CN 95:5). Theresulting peptides were trapped and desalted on a VanGuard pre-columnBEH C18 1.7 μm (2.1×5 mm (Waters Inc.)). Subsequently, the valves wereswitched to place the pre-column inline with the analytical column,UPLC-BEH(C18 1.7 μm (2.1×100 mm (Waters Inc.)), and the peptidesseparated using a 9 min gradient of 15-35% B delivered at 200 μl/minfrom an AQUITY UPLC system (Waters Inc.). The mobile phases consisted ofA: 0.1% formic acid and B: 0.1% formic acid in CH₃CN. The ESI MS data,and the separate data dependent MS/MS acquisitions (CID) and elevatedenergy (MS^(E)) experiments were acquired in positive ion mode using aQ-TOF Premier MS (Waters Inc.). Leucine-enkephalin was used as the lockmass ([M+H]⁺ ion at m. 556.2771) and data was collected in continuummode (For further description of the set-up, see Andersen and Faber,Int. J. Mass Spec., 302, 139-148(2011)).

Data Analysis

Peptic peptides were identified in separate experiments using standardCID MS/MS or MS^(E) methods (Waters Inc.). MS^(E) data were processedusing BiopharmaLynx 1.2 (version 017). CID data-dependent MS/MSacquisition was analyzed using the MassLynx software and in-house MASCOTdatabase.

HX-MS raw data files were subjected to continuous lock mass-correction.Data analysis, i.e., centroid determination of deuterated peptides andplotting of in-exchange curves, was performed using prototype customsoftware (HDX browser, Waters Inc.) and HX-Express ((Version Beta): Weiset al., J. Am. Soc. Mass Spectrom. 17, 1700 (2006)). All data were alsovisually evaluated to ensure only resolved peptide isotopic envelopeswere subjected to analysis.

Epitope Mapping Experiment

Amide hydrogen/deuterium exchange (HX) was initiated by a 6-8 folddilution of hTREM-1 in the presence or absence of mAb into thecorresponding deuterated buffer (i.e. PBS prepared in D20, 96% 20 final,pH 7.4 (uncorrected value)). All HX reactions were carried out at 20° C.and contained 4 μM hTREM-1 in the absence or presence of 4 μM mAb thusgiving a 2 fold molar excess of mAb binding sites. At appropriate timeintervals ranging from 10 sec to 10000 sec, 50 μl aliquots of the HXreaction were quenched by 50 μl ice-cold quenching buffer (1.35M TCEP)resulting in a final pH of 2.5 (uncorrected value),

Results and Discussion

This experiment maps the epitopes of mAbs 0023, 0024, 0025, 0026 and thecommercial mAbs MAB1278 (RnD Systems) and Clone26 (Biolegend) onhTREM-1. The HX time-course of 43 peptides, covering 94% of the primarysequence of hTREM-1, were monitored in the absence or presence of theeight different mAbs for 10 to 10000 sec.

Exchange protection observed in the early time-points, e.g. <300 sec,relate to surface exposed amide protons and thus also relate to proteininterfaces. In contrast, effects observed late in the time course arerelated to slow exchanging amide hydrogens and thus related to thestructural core of the protein. Therefore, epitope effects appear in theearly time points whereas structural stabilization effects will manifestas exchange reduction in late time points (Garcia, Pantazatos andVillareal. Assay and Drug Dev Tech. 2, 81 (2004); Mandell, Falick andKomives, Proc. Natl. Acad. Sci. USA, 95, 14705 (1998)).

The observed exchange pattern in the early timepoints in the presence orabsence of a given mAb can be divided into two different groups: Onegroup of peptides display an exchange pattern that is unaffected by mAbbinding. In contrast, another group of peptides in hTREM-1 showprotection from exchange upon mAb binding. For example at 100 secexchange with D₂O, approx than 2 amides are protected from exchange inthe region Y111-F126 of mAb 0023. Regions displaying such protectioneffects are assigned to the epitope region.

Epitope Mapping of mAbs 0123 and 0026

mAbs 0023 and 0026 both induce identical alterations in the exchangeprofile of hTREM-1 and will be described together here. The regionsdisplaying protection upon 0023/0026 binding encompass peptides coveringresidues T22-L96 and Y111-D127. However, by comparing the relativeamounts of exchange protection within each peptide upon binding mAb0023/0026 and the lack of epitope effects in peptides T25-F48, R84-Q112and peptides starting at P118, the epitope can be narrowed to residuesA21-E26, A49-I85 and C113-P119. Although distant in sequence, theseregions are close in the 3D structure of hTREM-1.

Epitope Mapping of mAb 0024 and Biolegend Clone 26

mAb 0024 and Clone26 from Biolegend both induce identical alterations inthe exchange profile of hTREM-1 and will be described together here. Theregions displaying protection upon mAb 0024 binding encompass peptidescovering residues V101-Q112. By comparing the relative amounts ofexchange protection within each peptide upon binding mAb 0024 and thelack of epitope effects in surrounding peptides, the epitope can benarrowed to residues Q104-Q112 (FIG. 7B).

Epitope Mapping of NNC mAb 0025

The regions displaying protection upon 0025 binding encompass peptidescovering residues D38-M63, T70-L96 and Y111-D127 (FIG. 7C). However, bycomparing the relative amounts of exchange protection within eachpeptide upon binding 0254-0025 and the lack of epitope effects inpeptides in surrounding regions, the epitope can be narrowed to residuesV39-Q56, T70-85 and C113-P119. Although distant in sequence, theseregions are close in the 3D structure of hTREM-1 (FIG. 8).

Eptiope Mapping of MAB1278

The regions displaying protection upon MAB1278 binding encompasspeptides covering residues T70-L96 and V101-Q112 (FIG. 7D). However, bycomparing the relative amounts of exchange protection within eachpeptide upon binding MAB1278 and the lack of epitope effects in peptidesin surrounding regions, the epitope can be narrowed to residues T70-185and Q104-Q112. Although distant in sequence, these regions are close inthe 3D structure of hTREM-1.

The structural position of the epitopes of mAbs 0023/0026 and mAb 0025are shown in FIG. 7A. The epitope of mAbs 0023 and 0026 seems to resideprimarily in β-sheets in the dimer interface of the hTREM-1 crystalstructure dimer. The antagonism of these mAbs could be a result ofpreventing hTREM-1 dimerisation and thus signalling.

Example 8: Determination of the Interaction Interface Between TREM-1 andmAb 0170

Epitopes were mapped on both recombinant human and cynomolgus monkeyTREM-1 (hTREM-1 and cTREM-1, respectively). The hTREM-1 construct usedin this example comprises the residues M1-H140 (SEQ ID NO: 18) and thecTREM-1 construct comprises the residues M1-R180 of (SEQ ID NO: 12) withsix histidine residues added to the C-terminus and using the amino acidnumbering from wild-type hTREM-1. Throughout this example the aminoacids of cTREM-1 are numbered according to the analogous residue inhTREM-1, as illustrated in FIG. 11. The numbering used in this examplecan be converted to the numbering in SEQ ID NO: 12 by subtracting 19 ifthe residue number is 58 or less and by subtracting 20 if the residuenumber is 60 or greater. As an example, the residue number E46 oncTREM-1 in this example corresponds to residue (46−19=27) E27 in SEQ IDNO: 12. The residue number on L96 on cTREM-1 in this example correspondsto residue (96−20=76) L76 in SEQ ID NO. 12.

Solutions of TREM-1, alone or in the presence of mAb 0170, were diluted25-fold in 97% deuterated hepes buffer (20 mM hepes, 150 mM sodiumchloride, pH 7.4) Non-deuterated controls were prepared by diluting intoprotiated hepes buffer. The hydrogen exchange experiments were performedon a waters HDX nanoAcquity ultra-high performance liquid chromatography(UPLC) system (Waters Corporation, Milford, Mass., USA) which includedthe HD-x PAL auto sampler (LEAP Technologies Inc., Carrboro, N.C., USA)for automated sample preparation. The LC tubing, pre- and analyticalcolumns and switching valves were located in a chamber cooled to 0.3° C.The trypsin digestion column was stored at 15° C. Hydrogen exchangereactions were performed at 20° C. Mass analysis was performed onlineusing a Waters SYNAPT G2 HDMS mass spectrometer.

A volume containing 100 pmol of human or cynomolgus TREM-1 (1.54-1.98μl) with or without 120 pmol mAb 0170 was diluted into deuterated hepesbuffer to a final volume of 50 μl. At the appropriate time intervals theentire volume was transferred to and quenched in 50 μl 1.35 mMTris(2-carboxyethyl)phosphine adjusted to pH 2.4 and held at 3° C. 99 μlof the quenched solution was immediately injected and passed over aPorozyme immobilised pepsin column (2.1 mm×30 mm)(Applied Biosystems,Life Technologies Corporation, Carlsbad, Calif., USA) and trapped on aWaters VanGuard BEH C18 1.7 μm (2.1 mm×5 mm) column at 100 μl/minflowrate using a 5% (vol/vol) methanol and 0.1% formic acid mobile fase.The peptides were separated on a Waters UPLC BEH C18 1.7 μm (1.0 mm×100mm) column using a 10-40% acetonitrile gradient containing 0.1% formicacid at a 40 μl/min flow-rate.

The mass spectrometer was operated in positive ion mode with ionmobility separation enabled. The electrospray conditions were 3.2 kVcapillary, 25 V sample cone, and 4 V extraction cone offsets, 850 ml/minflow of nitrogen desolvation gas heated to 350° C. and 50 ml/min conegas flow. The source block was heated to 120° C. Lock-mass correctiondata was acquired using the I+ ion of Leucine-enkephalin (m/z 556.2771)as reference compound and applied during data analysis. For peptideidentification MS^(E)-type experiments using trap collision offsets of 6V (low-energy) and 50 V (elevated energy) were performed. Deuteratedsamples were analysed using the 6 V low energy trap collision offsetonly. For further details see Andersen, M. D., Faber, J. H., Int. J.Mass Spectrom. (2011), 302, 139-148.

The MS^(E)-data was analysed using Waters ProteinLynx Global Server 2.5and peptides of hTREM-1 were identified that covered 80% of the proteinsequence (Table 3) and peptides of cTREM-1 were identified that covered100% of the protein sequence (Table 4). The HX-MS data files wereanalysed using Waters DynamX 1.0 that automatically applies lock-masscorrection and determines the degree of deuterium incorporation in eachpeptide. In addition, all data was manually inspected to ensure correctpeak assignment and calculation of deuterium incorporation

Results

A list of the peptides and their exchange patterns is provided in Table3.

When mAb 0170 bound hTREM-1, protection from exchange was observed inpeptides covering the sequence from A21 to L96 and the epitope wasconsequently determined to be within this region. When taking intoaccount peptides that show no protection from exchange upon binding ofmAb 0170, the epitope could be narrowed to the regions D38-F48. Theregion from R84-L96 showed little to no exchange in the presence or theabsence of mAb 0170 and it was not possible to conclude whether thisregion was part of the mAb 0170 binding epitope. The peptide K47-A68didn't show protection from exchange upon binding of mAb 0170, but thepeptide T44-C69 was protected when mAb 0170 was bound. The first tworesidues of a peptide back-exchanges quickly and exchange informationfor those residues is lost. It was concluded that at least one of theresidues E46, K47, and F48 was important for the binding of mAb 0170.

TABLE 3 Results from HXMS epitope mapping of mAb 0170 on human TREM-1Peptide mAb 0170 A21-L37 N A21-D38 N A21-V39 N A21-C69 EX T22-D38 ND38-Q56 EX D38-M63 EX D38-L67 EX D38-A68 EX D38-C69 EX V39-A68 EXV39-C69 EX D42-C69 EX T44-C69 EX K47-A68 N A49-C69 N I57-A68 N I57-C69 NI57-L87 N L67-L87 N A68-E88 N A68-L96 N C69-L87 N C69-L96 N T70-L87 NT70-E88 N T70-L96 N R84-L96 LE E88-L96 LE Q104-L110 N Y111-M124 NY111-F126 N Y111-D127 N C113-F126 N V114-F126 N V114-D127 N I115-F126 NI115-D127 N EX: Epitope region indicated by hydrogen exchange protectionupon antibody binding. (Exhange difference (EX) > 0.8 deuterons) W: Weakexchange due to structural effects (0.1 < EX < 0.8). N: No protectionfrom exchange upon antibody binding. (EX < 0.1) LE: Low intrinsicexchangemAb 0170 Epitope on cTREM-1

A list of the peptides and their exchange patterns is given in Table 4.

When mAb 0170 bound to cTREM-1, protection from exchange was observed inpeptides covering the sequence from E38 to A68 and the epitope wasconsequently determined to be within this region. When taking intoaccount peptides that shown no protection from exchange upon binding ofmAb 0170, the epitope could be narrowed to the regions E38-L45. Thisepitope corresponded well with the mAb 0170 epitope on hTREM-1 but wastruncated by three residues. The peptide C44-T69 in hTREM-1 wasprotected upon binding of mAb 0170, but the peptides A44-L67 and A44-A68that cover the corresponding sequence in cTREM-1 were not protected.Thus, whereas at least one of the residues 46, K47, and F48 in hTREM-1contributed to the binding epitope the corresponding residues E46, K47,and Y48 were not involved in binding of mAb 0170 to cTREM-1.

TABLE 4 HXMS epitope mapping of mAb 0170 on cynomolgus TREM-1 PeptidemAb 0170 T21-L37 W L24-L37 W T25-L37 W T25-E38 W E38-L67 EX E38-A68 EXV39-L67 EX V39-A68 EX K40-L67 EX A44-L67 W A44-A68 W E46-L67 N K47-L67 NA68-L87 W A68-L96 W A68-Q97 W K69-L87 W K69-L96 W V82-L96 W E88-L96 LEE88-Q97 LE Q97-L103 N Q104-L110 N Y111-C130 W Y111-L131 W V114-L131 WI115-C130 W I115-L131 W L131-T180 W L131-V182 W V132-T180 W V132-V182 WY152-T180 W V181-E189 N V181-H206 W I190-H206 N T193-H206 N V195-H206 NT196- H206 N D197-H206 N EX: Epitope region indicated by hydrogenexchange protection upon antibody binding. Exhange difference (EX) > 0.8deuterons) W: Weak exchange due to structural effects (0.1 < EX < 0.8).N: No protection from exchange upon antibody binding. (EX < 0.1) LE: Lowintrinsic exchange

Example 9: Study of Interaction Kinetics for Anti TREM-1 Antibodies toHuman and Cynomolgus TREM-1 by Surface Plasmon Resonance (SPR)

Binding studies were performed on a ProteOn Analyzer (BioRad) thatmeasures molecular interactions in real time through surface plasmonresonance. Experiments were run at 25° C. and the samples were stored at15° C. in the sample compartment. The signal (RU, response units)reported by the ProteOn is directly correlated to the mass on theindividual sensor chip surfaces in six parallel flow cells. Anti-humanFc monoclonal or anti-murine Fc polyclonal antibody from Biacore humanor mouse Fc capture kits were immobilized in horizontal direction ontoflow cells of a GLM sensor chip according to the manufacturer'sinstructions. The final immobilization level of capture antibody wasapproximately 2600-6000 RU in different experiments. Capture of purifiedmonoclonal mouse or recombinant expressed anti-hTREM-1 antibodies wasconducted by diluting the antibodies to 5-10 nM into running buffer (10mM Hepes 0.15 M NaCl, 5 mM EDTA, 0.05% surfactant P20, pH 7.4) andinjected in vertical direction at 30 μl/min for 60 s, creating referenceinterspots adjacent to all flow cells with only anti-Fc antibodyimmobilized. This typically resulted in final capture levels of testantibodies of approximately 100-300 RU and Rmax values of analyte of30-90 RU. Binding of hTREM-1 or cTREM-1 proteins was conducted byinjecting analyte (antigen) over all flow cells in horizontal directionto allow for comparative analyses of binding to different capturedanti-TREM-1 antibodies relative to binding to the reference interspot.hTREM-1 or cTREM-1 proteins was diluted serially 1:3 to 1.2-100 nM orinto running buffer, injected at 100 μl/min for 250 s and allowed todissociate for 600 s. The GLM surface was regenerated after eachinjection cycle of analyte via two 18 s injections of 10 mM Glycine, pH1.7 and 50 mM NaOH at 100 μl/min. This regeneration step removed theanti-TREM-1 antibody and any bound TREM-1 protein from the immobilizedcapture antibody surface, and allowed for the subsequent binding of thenext interaction sample pair. The regeneration procedure did not removethe directly immobilized anti-Fc capture antibody from the chip surface.

Binding affinity between antibodies and the antigen was quantified bydetermination of the equilibrium dissociation constant (K_(D))determined by measurement of the kinetics of complex formation anddissociation. The rate constants corresponding to the association andthe dissociation of a monovalent complex such as k_(a) (associationrate) and k_(d) (dissociation rate) were retrieved by fitting data to1:1 Langmuir model using the ProteOn evaluation software for dataanalysis. K_(D) is related to k_(a) and k_(d) through the equationK_(D)=k_(d)/k_(a).

Binding curves were processed by double referencing (subtraction ofreference surface signals as well as blank buffer injections overcaptured anti-TREM-1 antibodies) prior to data analysis. This allowedcorrection for instrument noise, bulk shift and drift during sampleinjections.

TABLE 5 Results from measurements of binding constants ka (associationrate), kd (dissociation rate) and KD (equilibrium dissociation constant)for the interaction of human TREM-1 to different anti-TREM-1 monoclonalantibodies. mAb Clone Origin ka (1/Ms) kd (1/s) KD (M) mAb 5F8A1Hybridoma 1.0E+05 2.2E−04 2.1E−09 0023 mAb 5F28A1 Hybridoma 7.8E+043.6E−04 4.6E−09 0024 mAb 5F19A1 Hybridoma 8.8E+05 8.5E−04 9.7E−10 0025mAb 5F27A1 Hybridoma 6.2E+04 9.7E−05 1.5E−09 0026 mAb 5F17A1B2 Hybridoma1.2E+05 4.2E−04 3.6E−09 0030 mAb 13F6A1 Hybridoma 1.2E+06 3.7E−033.1E−09 0031 mAb 13F10A1 Hybridoma 1.1E+06 2.1E−03 1.9E−09 0032 mAb9F11A1 Hybridoma 7.8E+04 1.1E−03 1.5E−08 0033 mAb 14F17A1 Hybridoma1.9E+05 1.9E−04 1.0E−09 0034 mAb 5F8A1 on Recombinant 8.9E+04 1.6E−041.8E−09 0039 mIgG2aa mAb 5F8A1 on Recombinant 9.3E+04 2.0E−04 2.2E−090040 mIgG1 mAb 5F27 on Recombinant 7.9E+04 1.8E−04 2.3E−09 0041 mIgG2aamAb 5F27A1 on Recombinant 8.5E+04 2.5E−04 2.9E−09 0042 mIgG1 mAb 14F69A1Hybridoma 1.5E+06 2.9E−04 2.0E−10 0044 mAb 13F14A1 Hybridoma 1.3E+063.2E−03 2.5E−09 0045 mAb 14F70A1 Hybridoma 9.3E+05 1.9E−04 2.1E−10 0046mAb 14F11A1B1 Hybridoma 1.7E+06 3.9E−03 2.4E−09 0048 mAb 14F86A1Hybridoma 8.8E+05 1.3E−03 1.5E−09 0049 mAb 5F24 HC645/ Recombinant6.8E+05 3.0E−03 4.5E−09 0051 LC647 mAb 14F128A1 Hybridoma 1.6E+063.8E−03 2.4E−09 0054 mAb 14F113/14F69 Recombinant 1.7E+06 5.2E−043.2E−10 0059 mAb 14F116A1B1 Hybridoma 1.0E+06 1.4E−03 1.4E−09 0063 mAb14F20A1B1 Hybridoma 8.9E+05 1.3E−03 1.5E−09 0064 mAb 14F11A1B1Recombinant 2.2E+06 3.9E−03 2.0E−09 0067 mAb 14F123/14F11 Recombinant2.7E+06 3.9E−03 1.7E−09 0068 mAb 14F113/14F69 Recombinant 2.1E+064.3E−04 2.5E−10 0070 mAb 14F1062A2 Hybridoma 9.8E+05 1.4E−03 1.5E−090078 mAb 14F29A1B1 Hybridoma 2.6E+05 1.2E−03 4.6E−09 0079 mAb 14F17A1B1Hybridoma 2.7E+05 1.9E−04 7.1E−10 0080 mAb 14F116A1B1 Recombinant1.0E+06 1.7E−03 1.7E−09 0083 mAb 14F113 fully Recombinant 2.1E+061.5E−03 7.7E−10 0090 humanized mAb 14F113HC/ Recombinant 1.8E+06 2.4E−021.6E−08 0115 14F128LC mixed and humanized mAb 14F113/14F69 Recombinant2.2E+06 1.5E−03 6.7E−10 0120 hz variant HC A78L mAb 14F113/14F69Recombinant 2.8E+06 1.7E−03 6.3E−10 0121 hz variant HC T93V mAb14F113/14F69 Recombinant 2.5E+06 1.4E−03 6.0E−10 0122 hz variant LC M4LmAb 14F113/14F69 Recombinant 2.0E+06 1.2E−03 6.1E−10 0124 hz variant LCG68R mAb 14F113/14F69 Recombinant 2.9E+06 5.4E−04 1.9E−10 0170 hzvariant LC Q98I

TABLE 6 Results from measurements of binding constants ka (associationrate), kd (dissociation rate) and KD (equilibrium dissociation constant)for the interaction of cynomolgus TREM-1 to different anti-TREM-1monoclonal antibodies. mAb Clone Origin ka (1/(Ms) kd (1/s) KD (M) mAb14F11A1B1 Hybridoma 1.4E+05 1.7E−04 1.2E−09 0048 mAb 14F113A1B1C1Recombinant 1.8E+05 1.2E−04 6.7E−10 0059 mAb 14F11A1B1 Recombinant3.8E+05 1.7E−03 4.7E−09 0067 mAb 14F128/14F11 Recombinant 2.8E+053.4E−03 1.2E−08 0068 mAb 14F116A1B1 Recombinant No No No 0083 bindingbinding binding mAb 14F113 fully Recombinant 1.4E+05 3.0E−04 2.1E−090090 humanized mAb 14F113 hz vari- Recombinant 1.3E+05 1.3E−04 9.4E−100121 ant HC T93V mAb 14F113 hz vari- Recombinant 1.7E+05 1.6E−04 9.7E−100122 ang LC M4L mAb 14F113 hz vari- Recombinant 1.7E+05 2.0E−04 1.1E−090124 ant LC G68R mAb 14F113 hz vari- Recombinant 2.5E+05 8.9E−04 3.6E−090170 ant LC Q98I

Example 10: Humanisation of the Blocking TREM-1 mAb 14F69

The variable regions of two lead antibodies were obtained from cloningof hybridomas 14F128A1 and 14F113A1B1C1. Both antibodies were clonedusing the SMARTER-RACE technique (Clontech). The humanization effort wasperformed as an iterative loop where CDR grafted antibodies were firstaffinity evaluated and then re-engineered to include more backmutationsuntil an acceptable affinity was retained, using hybridoma purifiedantibodies as a benchmark. The CDR grafted antibodies were designed insilico and ordered from a commercial vendor (www.genscript.com).Subsequent re-engineering of antibodies was performed using sitedirected mutagenesis (Stratagene). All antibodies were expressed inHEK293-6E cells in preparation for affinity testing. Below is adescription of the main considerations for selection of appropriatehuman germline and test of backmutations. All numbering of variableregions used in this example refers to the Kabat numbering scheme.

>m14F128A1_H (CDRs marked with bold and underlined) (SEQ ID NO: 8)

>m14F128A1_L (CDRs marked with bold) (SEQ ID NO: 9)

>m14F113A1B1C1_H (CDRs marked with bold) (SEQ ID NO: 10)

>m14F113A1B1C1_L (CDRs marked with bold) (SEQ ID NO: 11)

From an analysis of the sequences, the CDRs for m14F128A1 according toKabats definition are:

>CDR_H1

TYAMH

>CDR_H2

RIRTKS [N/S] NYATYY [V/A] DSVKD

>CDR_H3

DMG [I/A] RRQFAY

>CDR_L1

RASESVD [S/T] F [G/D] [I/Y] SF [M/L] H

>CDR_L2

RASNLES

>CDR_L3

QQSNEDPYT

With the differences between m14F128A1 and m4F113A1B1C1 given as[m14F128A1/m14F113A1B1C1].

A 3D model of m14F128A 1 was build using standard techniques in MOE[available from www.chemcomp.com] and all residues within 4.5 Å of theeffective CDR regions (VH: 31-35B, 50-58, 95-102; VL: 24-34, 50-56,89-97) were defined as mask residues. Mask residues are all potentiallyimportant for sustaining the binding in the CDRs.

The mask residues included positions 1-2, 4, 27-37, 47, 49-59, 69, 71,73, 78, 92-103 for the heavy chain and positions 1-5, 7, 23-36, 46,48-56, 58, 62, 67-71, 88-98 for the light chain.

Using germline searches of m14F128A1 and manual inspection, VH3_73 andJH4 were identified as being an appropriate human germline combinationfor the heavy chain and VKIV_B3 and JK2 were identified as theappropriate human germline combination for the light chain.

>VH3_13/JH4 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQASGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR/YFDYWGQGTLVTVSS >VKIV_B3/JK2DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST P/YTFGQGTKLEIKR

Humanisation was then performed with the following rules:

-   -   Residues outside the mask were taken as human.    -   Residues inside the mask and inside the Kabat CDR were taken as        murine.        -   Residues inside the mask and outside the Kabat CDR with            mouse/germline consensus were taken as the consensus            sequence.        -   Residues inside the mask and outside the Kabat CDR with            mouse/germline difference were subject to potential back            mutations.

Grafting the effective CDR regions of m14F128A1 into the germlinesformed the basic humanisation construct of m14F128A1, hz14F128A1.

>hz14F128A1_H EVQLVESGGGLVQPGGSLKLSCAASGFTFS TYAMH WVRQASGKGLEWVG RIRTKSNNYATYYAASVKG RFTISPDDSKNTAYLQMNSLKTEDTAVYYCTR DMGIRRQFAYWGQGTLVTVSS >hz14F128A1_L DIVMTQSPDSLAVSLGERATINC RASESVDSFGISFMHWYQQKPGQPPKL LIY RASNLES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQSNEDPY TFGQGTKLEIK >CDR_H1 TYAMH >CDR_H2 RIRTKSNYATYYAASVKG >CDR_H3DMGIRRQFAY >CDR_L1 RASESVDSFGISFMH >CDR_L1 RASNLES >CDR_L3 QQSNEDPYT

The only differences compared to the murine CDRs were in CDR_H2 (shownin bold). Any discrepancy between m14F128A and hz14F128A1 in a maskresidue will create a potential backmutation and the list includes:

hz14F128A1_H: S30N, G49A, A78L, V93T

hz14F128A1_L: M4L, M58I, G68R

Furthermore, the close homology of m14F128A1 and m14F113A1B1C1 was usedto suggest residues that could impact the affinity of hz14F128A1.

hz14F128A1_H: N53S, 198Q

hz14F128A1_L: S27D_T, G29D, I30Y, M33L

In order to investigate all potentially humanised mAbs all combinationsof the above mutants were produced and tested.

The final humanised anti hTREM1 antibody (mAb 0170) derived fromhybridoma 14F113 contains one LC framework-backmutation (M4L) and one HCCDR3 mutation (Q98I). The mutation in HC CDR3 was introduced based on anaffinity-synergy-study with a highly homologous antibody named 14F128.The rationale for including both mutations is described below.

Affinity-Synergy Study of Antibody 14F128 and 14F113

The hybridoma antibodies 14F128 and 14F113 are highly homologous andderived from the same somatic recombination event. The two antibodiescompete in hTREM1 binding with 14F113 having the highest affinity. Intotal the CDR grafted versions of the two antibodies differ in their CDRcompositions by only six amino acids (four in LC CDR, and two in HCCDR). The six mutations, when comparing 14F113 to 14F128, are LCT27_(d)S, D29G, Y30I, L33M and HC S54N, Q98I. Although CDR grafted14F128 had an affinity inferior to CDR grafted 14F113 it wasinvestigated if a beneficial affinity effect from one or more of the sixmutations was suppressed by the overall effect when all six mutationswere present. All six mutations (except HC S54N) were thereforeindividually introduced in the CDR grafted 14F113 antibody and theantibodies were ranked by affinity. Two mutations (LC L33M and HC Q98I)were individually capable of improving the affinity of CDR grafted14F113. A mutation of the HC at position Q98I gave rise to aparticularly good affinity of the resultant antibody (mAb 0170).

Framework Backmutation Affinity Analysis

The mouse version of the 14F113 antibody had seven mutations that werepotentially necessary to include as backmutations during humanisation.The potential backmutations in the HC and LC were S30N, G49A, A78L andT93V M4L, V58I, G68R, respectively. The seven backmutations wereintroduced individually in CDR grafted 14F113 and then ranked byaffinity. Although several of the mutations were capable of improvingaffinity, only LC mutation M4L was selected for mAb 0170 The decision toinclude mutations was balanced against expression titer (HEK293 6E),affinity, and the total number of mutations.

Example 11: Study of Interaction Kinetics for TREM-1 Antibodies tohTREM-1 by Surface Plasmon Resonance (SPR): Comparison Between mAb 0170and Commercially Available TREM-1 Antibodies

Binding studies were performed on a ProteOn Analyzer (BioRad) thatmeasures molecular interactions in real time through surface plasmonresonance. Experiments were run at 25° C. and the samples were stored at15° C. in the sample compartment. The signal (RU, response units)reported by the ProteOn is directly correlated to the mass on theindividual sensor chip surfaces in six parallel flow cells. Commerciallyavailable antibodies included were Biolegend #314907, Biolegend #316102(Biolegend, USA), Hycult Biotech H11M2252 (Hycult Biotech, Netherlands),R&D #MAB1278 (R&D systems. United Kingdom), SC98Z12 (Santa CruzBiotechnology, USA), Sigma #WH0054210m4, Sigma #SAB1405121(Sigma-Aldrich Danmark A/S)

Anti-human Fc monoclonal or anti-murine Fc polyclonal antibody fromBiacore human or mouse Fc capture kits were immobilized in horizontaldirection onto flow cells of a GLM sensor chip according to themanufacturer's instructions. The final immobilization level of captureantibody was approximately 2600-6000 RU in different experiments.Capture of purified monoclonal mouse or recombinant expressed humanizedanti-hTREM-1 antibodies was conducted by diluting the antibodies to 5-10nM into running buffer (10 mM Hepes 0.15 M NaCl, 5 mM EDTA, 0.05%surfactant P20, pH 7.4) and injected in vertical direction at 30 μl/minfor 60 s, creating reference interspots adjacent to all flow cells withonly anti-Fc antibody immobilized. This typically resulted in finalcapture levels of test antibodies of approximately 100-300 RU and Rmaxvalues of analyte of 30-90 RU. Binding of hTREM-1 or cTREM-1 proteinswas conducted by injecting analyte over all flow cells in horizontaldirection to allow for comparative analyses of binding to differentcaptured anti-TREM-1 antibodies relative to binding to the referenceinterspot. hTREM-1 or cTREM-1 proteins was diluted serially 1:3 to1.2-100 nM or into running buffer, injected at 100 μl/min for 210 s andallowed to dissociate for 600 s. The GLM surface was regenerated aftereach injection cycle of analyte via two injections of 10 mM Glycine, pH1.7 and 50 mM NaOH at 100 μl/min. This regeneration step removed theanti-TREM-1 antibody and any bound TREM-1 protein from the immobilizedcapture antibody surface, and allowed for the subsequent binding of thenext interaction sample pair. The regeneration procedure did not removethe directly immobilized anti-Fc capture antibody from the chip surface.

Binding affinity between antibodies and the antigen was quantified bydetermination of the equilibrium dissociation constant (K_(D))determined by measurement of the kinetics of complex formation anddissociation. The rate constants corresponding to the association andthe dissociation of a monovalent complex such as k_(a) (associationrate) and k_(d) (dissociation rate) were retrieved by fitting data to1:1 Langmuir model using the ProteOn evaluation software 3.1.0.6 fordata analysis. K_(D) is related to k_(a) and k_(d) through the equationK_(D)=k_(d)/k_(a).

Binding curves were processed by double referencing (subtraction ofreference surface signals as well as blank buffer injections overcaptured anti-REM-1 antibodies) prior to data analysis. This allowedcorrection for instrument noise, bulk shift and drift during sampleinjections.

TABLE 7 Results from measurements of KD (equilibrium dissociationconstant) for the interaction of human and cynomolgus TREM-1 todifferent anti-TREM-1 monoclonal antibodies. human TREM-1 cyno TREM1Antibody KD (M) KD (M) mAb 0170 2E−10 3E−09 Biolegend #314907 9E−104E−09 Biolegend #316102 8E−10 No binding Hycult Biotech HM2252 3E−09 Nobinding R&D #MAB1278 8E−09 No binding SC98Z12 3E−08 No binding Sigma#WH0054210m4 21−08 No binding Sigma #SAB1405121 No binding No binding

Example 12: Competition Binding Studies of Anti-Human TREM-1 MonoclonalAntibodies by Surface Plasmon Resonance

SPR binding competition studies were performed with monoclonal mouse orrecombinant expressed humanised anti-hTREM-1 antibodies in order todiscriminate between different binding sites (epitopes). Commerciallyavailable antibodies included were Biolegend #4314907 (Biolegend, USA)and SC98Z12 (Santa Cruz Biotechnology, USA). Anti hTREM-1 monoclonalantibodies that compete for the same or an overlapping binding site(epitope) on the antigen are not able to bind simultaneously to theantigen and are therefore assigned to the same “bin”. Anti-TREM-1monoclonal antibodies that do not compete for the same or overlappingbinding site on the antigen are able to bind simultaneously and are thusassigned to different “bins”. Experiments were performed with soluble,human TREM-1 extracellular domain as antigen.

All studies were run at 25° C., and the samples were stored at 15° C. inthe sample compartment. Individual anti-TREM-1 monoclonal antibodies andan unrelated control monoclonal antibody were immobilised onto separateflow cells of a GLC sensor chip using a 1:1 mixture of 0.4 M EDAC[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] and 0.1 MSulfo-NHS [N-hydroxysulfosuccinimide]. Each antibody was diluted in 10mM sodium acetate pH 5.0 to a concentration of 25 or 10 (SC98Z12(80394)) μg/ml, and was immobilised to an individual flow cell at 30μl/min for 240 s. The antibodies were immobilised to flow cells L1-L6(including control). After immobilisation of the antibody, the activesites on the flow cell were blocked with 1 M ethanolamine.Immobilisations were performed with activation and deactivation in ahorizontal direction creating interspot reference points withoutimmobilised protein. The final immobilisation level of test antibodiesranged from approximately 1100 to 1300 RU in one experiment, except forone antibody (SC98Z12) where only 390 RU was immobilised. Recombinanthuman TREM-1 was diluted to 100 nM into running buffer (10 mM Hepes 0.15M NaCl, 5 mM EDTA, 0.05% surfactant P20, pH 7.4). The antigen wasinjected over immobilised antibodies in horizontal direction at 30μl/min for 300 s, allowing control of potential unspecific binding bothto interspot references and immobilised control antibodies resulting in150-600 RU captured TREM-1, except for to one antibody (SC98Z12) withlow immobilisation level where only 4 RU was captured.

Each antibody (the same ones that had been immobilised) was injectedover parallel flow cells in a horizontal direction to allow forcomparative analysis of binding to hTREM-1 captured by the primaryantibodies relative to binding to both the interspot references and theimmobilised control antibodies. Each competing antibody was diluted to100 nM and injected at 100 μl/min for 250 s. The GLC chip wasregenerated after each injection cycle of analyte via two 18 sinjections of 1M Formic acid pH 3.5, 3M MgCl2 and 50 mM NaOH at 100μl/min. This regeneration step removed the TREM-1 antigen and any boundsecondary antibody from the immobilised antibody surface, and allowedfor the subsequent binding of the next test sample. The regenerationprocedure did not remove the directly immobilised anti-TREM-1 testantibody (primary antibody) from the chip surface. Data analysis wasperformed with the ProteOn Manager™ 3.1.0.6 Software. Capture levelswere assessed to ensure that the regeneration step provided a consistentbinding surface throughout the sequence of injections. No significantnon-specific binding of human TREM-1 neither to the interspot controlsurfaces nor to immobilised control antibody was observed. Bindingcurves were processed by subtraction of interspot control surfacesignals. This allowed correction for instrument noise and bulk shiftduring sample injections.

The competition results were reported as either positive or negativebinding (Table 8). Positive (+) binding indicates that the competingantibody was capable of binding the hTREM-1 simultaneously with theprimary antibody (i.e. they do not compete), and the primary andcompeting antibodies were consequently assigned to different epitopebins. Negative binding indicates that the competing antibody was unableto bind the hTREM-1 simultaneously with the primary antibody (i.e. theydo compete), and the primary and competing antibodies were thus assignedto the same epitope bin. The response values in these experiments weresignificant and allowed for an unambiguous determination of epitope binsof the anti-TREM-1 monoclonal antibodies.

TABLE 8 Ability to bind (+) or to compete (−) for antibodies tested inSPR competition assay. SC98Z12 did not give high enough capture ofTREM-1 to evaluate as primary antibody (*). Primary Biolegend mAb mAbSecondary #314907 SC98Z12 0048 0170 Biolegend #314907 − * + + SC98Z12− * + + mAb 0048 + * − − mAb 0170 + * − −

MAb 0170 and mAb 0048 (purified from hybridoma 14F11, which is identicalto 14F128) were shown to compete for binding to human TREM-1. Biolegend#314907 and SC98Z12 did not compete with any of these for human TREM-1binding but competed with each other. These findings conclude that thefirst two (mAb 0048 and mAb 0170) belong to the same bin (Bin1) whileBiolegend #314907 and SC98Z12 belong to another bin (Bin2).

Example 13: Kinetic Analysis of the Interaction Between Fab 0011 and Fab0170 Binding to Mutated Versions of Human and Cynomolgus TREM-1

Interaction studies were performed by SPR to define differences in theepitopes for 0011 and 0170 anti-humanTREM-1 antibodies on human TREM-1.By comparing binding kinetics to human M-variants with introducedAlanine mutations in known epitopes, as well as partly“humanised”variants of cynomolgus TREM-1, the latter since only mAb 0170 crossreacts with cynomolgus TREM-1 amino acid residues unique for respectiveepitope were identified.

The hTREM-1 extracellular domain alanine mutant constructs and partlyhumanised cynomolgus mutant constructs used in this study are summarisedin Table 9. All constructs used were variants either of SEQ ID NO:19(cynomolgus TREM-1 variants) or SEQ ID NO: 20 (human TREM-1 variants)and included a C-terminal-cmyc²-His⁶ tag for capture in SPR bindingkinetics assay. Unless otherwise stated, sequences referred to in thisexample are numbered according to FIG. 11.

TABLE 9 TREM-1 variant Protein Mutations 0221 human TREM-1 Ala mut 1D38A 0222 human TREM-1 Ala mut 2 K40A 0223 human TREM-1 Ala mut 3 D42A0224 human TREM-1 Ala mut 4 T44A 0225 human TREM-1 Ala mut 5 E46A 0226human TREM-1 Ala mut 6 K47A 0227 human TREM-1 Ala mut 7 S50A 0228 humanTREM-1 Ala mut 8 R84A 0229 human TREM-1 Ala mut 9 Y90A 0230 human TREM-1Ala mut 10 H91A 0231 human TREM-1 Ala mut 11 D92A 0232 human TREM-1 Alamut 12 H93A 0233 human TREM-1 Ala mut 13 R97A 0234 human TREM-1 Ala mut14 R99A 0235 human TREM-1 Ala mut 15 D127A 0236 human TREM-1 Ala mut 16R128A 0237 human TREM-1 Ala mut 17 R130A 0238 cynomolgus TREM-1 wt —0239 cynomolgus TREM-1 “partly A44T, Y48F, N50S, humanised” R52Q, E75K,P91H 0240 cynomolgus TREM-1 “partly A44A, V48F, N50S, humanised withback mutation 1” R52Q, E75K, P91H 0241 cynomolgus TREM-1 “partly A44T,Y48Y, N50S, humanised with back mutation 2” R52Q, E75K, P91H 0242cynomolgus TREM-1 “partly A44T, Y48F, N50N, humanised with back mutation3” R52Q, E75K, P91H 0243 cynomolgus TREM-1 “partly A44T, Y48F, N50S,humanised with back mutation 4” R52R, E75K, P91H 0244 (SEQ cynomolgusTREM-1 “partly A44T, Y48F, N50S, ID NO: 14) humanised with back mutation5” R52Q, E75E, P91H 0245 (SEQ cynomolgus TREM-1 “partly A44T, Y48F,N50S, ID NO: 15) humanised with back mutation 6” R52Q, E75K, P91P 0247human TREM-1 wt —

Binding studies were performed on a ProteOn Analyzer that measuresmolecular interactions in real time through surface plasmon resonance.Experiments were run at 25° C. and the samples were stored at 15° C. inthe sample compartment. The signal (RU, response units) reported by theProteOn is directly correlated to the mass on the individual sensor chipsurfaces in six parallel flow cells. Anti-His monoclonal antibody wasimmobilised onto 6 parallel flow cells of a GLM sensor chip using a 1:1mixture of 0.4 MEDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodimidehydrochloride] and 0.1 M Sulfo-NHS [N-hydroxysulfosuccinimide]. Antibodywas diluted in 10 mM sodium acetate pH 5.0 to a concentration of 25μg/ml, and was immobilised onto individual flow cells at 30 μl/min for240 s. After immobilisation of the antibody, the active sites on theflow cells were blocked with 1 M ethanolamine. Immobilisation wasperformed with all steps in horizontal direction. The finalimmobilisation level of capture antibody was approximately 8000 RU inone experiment. Cell culture medium from HEK 293 cells expressing wildtype or different mutated variants of human or cynomolgus TREM-1 ECD wasdiluted 40-60 times in running buffer (10 mM HEPES, 150 mM NaCl, 3 mMEDTA, 0.05% surfactant P20, pH 7.4). TREM-1 proteins were injected overimmobilised anti-His capture antibody in the vertical direction at 30μl/min for 60 s. This resulted in 50-250 RU captured TREM-1 and createdinterspot references with only immobilised capture antibodies but nocaptured TREM-1 in the horizontal direction. Each Fab was injected overparallel flow cells in the horizontal direction to allow for kineticanalysis of binding to TREM-1 variants captured by anti-His antibody.Prior to injection each Fab was diluted to 0, 5.5 (in one experiment),16.7 and 50 nM in running buffer, and injected at 100 μl/min for 250 s(association time). The dissociation time following these injections wasmonitored for 10 minutes. The GLM chip was regenerated after eachinteraction cycle of TREM-1 and Fab via two 18 s injections of 10 mMGlycine and 50 mM NaOH at 100 μl/min. This regeneration step removed theTREM-1 variants and any bound Fab from the anti-His antibody surface,and allowed for the subsequent binding of the next interaction pair. Theregeneration procedure did not remove the directly immobilised anti-Hiscapture antibody from the chip surface.

In order to obtain kinetic data, such as ka (association rate), kd(dissociation rate) and KD (equilibrium dissociation constant), dataanalysis was performed using the ProteOn Manager™ 3.1.0.6 Software.Capture and binding levels of samples run in duplicates or triplicateswere assessed to ensure that the regeneration step provided a consistentbinding surface throughout the sequence of injections. No significantunspecific binding to interspot references with only immobilised captureantibody was observed. Binding curves were processed by subtraction ofinterspot control surface signals, as well as injection of runningbuffer. This allowed correction for instrument noise and bulk shiftduring sample injections.

The affinity of 0170 Fab and 0011 Fab to different TREM-1 ECD variantswhere compared to the affinity to wild type human or cynomolgus TREM-1ECD.

The level of binding 10 s after end of injection of Fab, normalised tolevel of captured TREM-1 variant, was also assessed in order to identifymutated versions with abrogated or very low binding. A decrease inaffinity combined with significantly lower normalised binding level canindicate a disrupted folding due to introduced mutations. It shouldhowever be noted that changes in kinetics will also affect this value.Each binding curve was therefore visually inspected for conclusion (datanot shown).

Results for human TREM-1 with alanine single mutations (Table 10) showthat two positions (T46 and D92) were unique in that the decreasedbinding more than two-fold to 0170 Fab.

TABLE 10 Affinity of 0170 Fab and 0011 Fab to human TREM-1 with alaninemutations relative to human TREM-1 wt. Binding level of 0170 Fab and0011 Fab 10 s after end of association, expressed as degree oftheoretical maximum binding level. Normalised binding (% KD relative to0247 Rmax theor) Construct Mutation 0170 Fab 0011 Fab 0170 Fab 0011 Fab0247 — 1 1 44 41 0221 D38A 0.7 15.0 29 5 0222 K40A 0.9 No binding 43 10223 D42A 132.9 26.9 13 3 0224 T44A 1.2 1.8 46 40 0225 E464 2.7 0.9 4444 0226 K47A 1.0 1.1 40 38 0227 S504 0.7 0.9 45 41 0228 R84A 0.5 1.3 158 0229 Y90A 0.5 2.9 40 31 0230 H91A 0.9 2.5 41 33 0231 D92A 9.2 1.1 3441 0232 H93A 36.4 5.2 22 29 0233 R97A 1.1 6.2 36 22 0234 R99A 0.8 1.0 1511 0235 D127A 1.1 0.8 43 41 0236 R128A 0.9 1.2 39 35 0237 R130A 1.0 0.835 34

mAb-0170 was, in contrast to mAb-0011, able to bind cynomolgus TREM-1.Humanisation of cynomolgus TREM-1 in the selected area did not result inregained affinity of 0177 compared to human TREM-1 (0247) indicatingthat other residues or combinations of residues are important for thedifferences in affinity for human and cynomolgus TREM-1.

0243 shows no binding to 0170 Fab or 0011 Fab. For this construct itcannot be excluded that mutations have affected the overall structureand therefore not be concluded if Q52 is involved in binding of thestudied Fabs to human TREM-1.

TABLE 11 Affinity of 0170 Fab and 0011 Fab to wt cynomolgus TREM-1 ECD,partly humanised cynomolgus TREM-1 ECD variants and wt human TREM-1 ECD.Binding level of 0170 Fab and 0011 Fab 10 s after end of association,expressed as degree of theoretical maximum binding level. Normalised KD(M) binding (% Rmax theor) Construct 0170 Fab 0011 Fab 0170 Fab 0011 Fabwt cTREM-1 1E−09 No binding 39 0 0239 2E−09 >1 uM 19 1 0240 2E−09 >1 uM28 0 0241 2E−09 >1 uM 26 2 0242 1E−09 >1 uM 21 1 0243 No binding Nobinding 5 1 0244 1E−09 No binding 7 0 0245 1E−09 No binding 34 0 02472E−10 1E−09 45 42

Example 14: mAb 0170 Efficiently Blocks TREM-1 Activation in theBWZ/hTREM-1 Reporter Cell Assay

The BWZ/hTREM-1 reporter cell assay described in Example 6 was used tocalculate the potency of mAb 0170 in blocking TREM-1. BWZ/hTREM-1reporter cells were stimulated with TREM-1 ligand complex and mAb 0170added at various concentrations. FIG. 9 shows a dose-dependent blockingof the TREM-1 signal resulting in total block of the signal atconcentrations higher than 0.2 ug/ml. The IC50 value was determined tobe 2.4 nM using the Graphpad Prism software, equation: log(inhibitor)vs. response—Variable slope.

Example 15: Commercially Available TREM-1 Antibodies do not Block TREM-1Activation in the BWZ/hTREM-1 Reporter Cell Assay

Multiple doses of antibody were included in the BWZ/hTREM-1 reportercell assay, as described previously. SAB1405121 (clone 3F5 from SigmaAldrich, St. Louis, Mo., USA), WH0054210M4 (clone 2E2 from SigmaAldrich, St. Louis, Mo., USA), sc-80394 (clone 98Z12 from Santa Cruz,Calif., USA), HM2252 (clone 611 from Hycult biotech, 5405 PB UDEN, TheNetherlands). 316102 (clone TREM-37 from Biolegend, San Diego, Calif.92121, USA), and 314907 (clone TREM-26 from Biolegend, San Diego, Calif.92121, USA), were not able to block the TREM-1 activity significantly,whereas mAb 0170 disclodes herein could block TREM-1 activity with morethan 99% at 0.3 ug/ml. Isotype controls had >95% remaining reactivityeven at 3 ug/ml.

TABLE 12 % % % % remaining remaining remaining remaining Anti TREM-1activity at 0 activity at activity at activity at clone μg/ml 0.003μg/ml 0.03 μg/ml 0.3 μg/ml SAB1405121 100 104 101 97 WHO054210M4 100 98104 86 sc-80394 100 120 134 131 HM2252 100 96 122 113 316102 100 98 10390 (0.09% azide) 314907 100 100 124 108 mAb 0170 100 96 80 0

Example 16: mAb 0170 Blocked Cynomolgus TREM-1

In order to test for functionality against TREM-1 from other species,mouse and cynomolgus monkey TREM-1 was transfected together with humanand mouse DAP12, respectively, to generate a reporter cell assay as theone for human. This was essentially done as described for the humansystem (Examples 1, 2 and 6) but replacing humanTREM-1 with full lengthmurine (m)TREM-1 (SEQ ID NO: 22) or full length cynomolgus monkey (c)TREM-1 (SEQ ID NO: 21). cDNA encoding cTREM-1 (SEQ ID NO: 22) wassynthesized at GeneArt and cloned into pHLEF38 (licensed from CMC ICOS),in the XhoI-XbaI orientation.TE alpha NFAT Luc cells was co-transfectedwith pHLEF38.cynoTrem1 and pNEF38 NFlag hDAP12 using 10 ug of eachplasmid and electroporated 8e6 cells in approx. 500 ul total volume (400ul growth medium and 100 ul DNA) using the BTX electoporator (260 V,1050 uF, 720 ohms; time constant was 23 msec). Cells were plated for 48hours in a 10 cm plate, in 10 ml of 50% conditioned medium and plateddirectly into selection at 8e3 cells/well of a flat bottomed 96W plates(5 plates) in 200 ul/well of 30% conditioned medium with 800 ug/ml G418and 0.5 mM L-histidinol. After 2 weeks of incubation, 40 single colonieswere identified using the Genetix Clone Select Imager.

The only commercially available TREM-1 antibody able to cross react withcynomolgus monkey TREM-1 was 314907 (clone TREM-26 from Biolegend, SanDiego, Calif. 92121, USA)(see Example 11). None of the commerciallyavailable antibodies tested in Example 15 were able to block thefunction of cynoTREM-1, not even the one that could bind to cynomolgusmonkey TREM-1.

TABLE 13 % remaining cyno TREM-1 activity [mAb amount] mAb 0170 314907  0 ug/ml 100.00 100.00 0.74 ug/ml 7.97 103.97   20 ug/ml 0 87.72

Likewise, a reporter cell line with mouse TREM-1 was generated. None ofthe antibodies able to bind to human TREM-1 or to cynomolgus and humanTREM-1 could cross-bind to mouseTREM-1. Thus, antibodies against mouseTREM-1 were generated essentially as described for generating humanTREM-1 antibodies but with mouse TREM-1 as the antigen. These antibodieswere screened for mouse TREM-1 binding and blocking function in themurine reporter gene assay. One such antibody (mAb 0174) was able tobind and block the mouse TREM-1 function.

Example 17: TNFalpha Release from M2 Macrophages that were Stimulated byPGLYRP-1 was Blocked by TREM-1 Antibodies

Those skilled in the art will recognize the value of establishing afreezer bank collection of primary cells from multiple donors thusproviding for convenient replication of experiments. In vitro derivedmacrophages were produced from peripheral blood monocytes as follows.Negatively enriched monocytes were isolated from a peripheral blood“leukopak” obtained from Research Blood Components (Brighton, Mass.,USA) using a Rosette Sep kit (cat. no. 15068) from Stem CellTechnologies (Vancouver, BC, Canada) following the manufactureinstructions. Isolated monocytes were suspended in 10% DMSO/FBS aliquotsof 50e6 cell/ml and gradually cooled to −80 C. To produce macrophagecells, one or more frozen vials of monocytes were rapidly thawed in a 37C water bath, diluted to 10 ml with growth media [RPMI 1640 (Gibco,Carlsbad Calif., USA) cat. no. 72400-047) with 10% FBS (FisherScientific cat no 03-600-511] and centrifuged 5 minutes at 250 g. Cellswere suspended to 2e6 cells/ml in growth media supplemented with 50ng/ml human MCSF (Gibco cat. no. PHC9501), placed into tissue culturetreated, petri style tissue culture plates and into a humidifiedincubator programmed to maintain a “hypoxic” atmosphere of 5% CO2, 2%O2. On the third day in culture, the cells were fed with the addition ofan equal volume of growth media supplemented with 50 ng/ml human MCSF.After 6 days in culture the monocytes had differentiated into M0macrophages. M0 cells were further differentiated by changing the mediato growth media supplemented with 50 ng/ml human IFNg (Gibco cat noPHC4031) for M1 macrophages or 40 ng/ml human IL-4 (Gibco cat noPHC0045) for M2 macrophages and returning to the incubator for anadditional 22 hours. On the seventh day, macrophages were suitablydifferentiated to be used in a bioassay. Briefly, macrophages wererecovered from the petri plates by washing with 1×PBS, followed by 5 mMEDTA in PBS. The plates were then returned to 37 C for 30 minutes andcells were “power washed” off the plate using a 10 ml syringe and 22Gneedle. Cells were then diluted into growth media, centrifuged at 250 gfor 5 minutes after which the cell pellet was suspended to a finalconcentration of 1e6/ml.

Macrophage cells prepared as above were used in bioassays wherecytokines such as TNF-alpha produced in response to stimulation of thecells with TREM-1 ligand were measured in the conditioned media byELISA. Such a bioassay was further utilized to measure blockade of suchTREM-1 ligand stimulation by TREM-1 specific antibodies. TREM ligand ornegative controls were prepared at 4× concentrations and 50microliters/well in growth media were added to 96 well microtiterdishes. Final concentrations of TREM-1 ligand consisted of 7.5 ng/mlrecombinant human PGLYRP1 (see Example 5) and 3 μg/ml PGN-BS (Invivogen,tlrl-pgnbs, SanDiego Calif., USA). Cells were cultured under humidifiedhypoxic conditions as described above for 22 hours after whichconditioned media was collected and TNF-alpha levels were measured byELISA, following manufacturer's instructions (R&D Systems, catalogueDY210, MN, USA). FIG. 10 shows that the TREM-1 antibodies decreaseTNFalpha release from these stimulated M2 macrophages.

Table 12, below, shows the IC50 values from such experiment indicatingthat the antibodies disclosed herein are very potent in blocking theTREM-1 dependent cytokine release.

TABLE 14 Antibody (mAb) R square IC50, ng/ml 0122 0,99 14 0170 0.99 120090 0.99 21 0115 0.88 92

Example 18: TNFalpha Release from Cynomolgus Macrophages can be Blockedby mAb 0170

Peripheral blood derived macrophage serve as an excellent in vitro modelin the study of innate immune modulation and activation. TREM-1 is knownto play a key role in controlling this process. The use of the non-humanprimate species Macaca fascicularis, commonly known as Cynomolgus monkeyis critical to understanding the in vivo effects of modulating TREM1signalling. In this example anti-TREM-1 antibodies are tested for theirability to block production of cytokines in cynomolgus M2 macrophagecultures.

Macrophage cells were generated as follows. Whole blood was harvestedfrom healthy male adult animals (SNBL, Everett Wash., USA) byvenipuncture using sodium heparin vacutainer tubes (Cat No 3664870,Bectin Dickinson Franklin Lakes N.J., USA). Whole blood was diluted 30%with PBS, then 30 ml was carefully layered on to 15 ml of Ficoll-Paque(Cat No 17-1440-03 GE Healthcare, Uppsala Sweden) prediluted to 96% withPBS in a 50 ml conical tube. After centrifugation: 30 min, roomtemperature, 400 g with low acceleration and no brake; peripheral bloodmononuclear cells (PBMC) were harvested from the Ficoll/plasmainterphase, diluted 3× with PBS and centrifuged 7 minutes, roomtemperature, 200 g. Supernatant containing contaminating platelets wasaspirated and discarded and the cell pellet was resuspended in 30 ml ofPBS+0.2% FBS (fetal bovine serum). This cell wash process was repeated 2additional cycles after which the PBMC cell pellet was resuspended inRPMI culture media (Cat No. 61870-036, Life Technologies, Grand IslandN.Y., USA) plus 10% FBS to 2E6 cells/ml and dispensed onto 15 cm petridishes (Cat No 430599 Corning, Tewksbury Mass., USA) at 20 ml/dish.Monocytes were allowed to adhere to plastic by incubating overnight at37 C, 5% CO₂, 100% humidity after which non adherent cells were removedby gently swirling and rocking the plates for 20 seconds followed byaspiration. 20 ml of fresh growth media supplemented with 50 ng/ml hMCSF(Cat No. PHC9501, Life Technologies, Grand Island N.Y., USA) was addedto each plate and placed into hypoxic culture conditions of 37 C, 5%CO₂, 2% O₂, 100% humidity for 7 days. On the third day in culture, thecells were fed with the addition of an equal volume of growth mediasupplemented with 50 ng/ml human MCSF. After 6 days in culture themonocytes had differentiated into M0 macrophages. M0 cells were furtherdifferentiated by changing the media to growth media supplemented with50 ng/ml human IFNg (Gibco cat no PHC4031) for M1 macrophages or 40ng/ml human IL-4 (Cat No. PHC0045, Life Technologies, Grand Island N.Y.,USA) for M2 macrophages and returning to the incubator for an additional22 hours. On the seventh day, macrophages were suitably differentiatedto be used in a bioassay. Briefly, macrophages were recovered from thepetri plates by washing with 1×PBS, followed by 5 mM EDTA in PBS. Theplates were then returned to 37 C for 30 minutes and cells were “powerwashed” off the plate using a 10 ml syringe and 22G needle. Cells werethen diluted into growth media, centrifuged at 250 g for 5 minutes afterwhich the cell pellet was suspended to a final concentration of 1e6/ml.

Macrophage cells prepared as above were used in bioassays wherecytokines such as TNF-alpha produced in response to stimulation of thecells with TREM-1 ligand were measured in the conditioned media byELISA. Such a bioassay was further utilized to measure blockade of suchTREM-1 ligand stimulation by TREM-1 specific antibodies. TREM-1 ligandor negative controls were prepared at 4× concentrations and 50microliters/well in growth media were added to 96 well microtiterdishes. Final concentrations of TREM-1 ligand consisted of 7.5 ng/mlrecombinant human PGLYRP1 (generated as described in Example 5) and 3μg/ml PGN-BS (Cat No. tlrl-pgnbs, Invivogen SanDiego Calif., USA).Antibodies were added in 50 microliters/well of growth media followed bycells in 50 microliters/well of macrophage. Cells were cultured underhumidified hypoxic conditions as described above for 22 hours afterwhich conditioned media was collected and TNF-alpha levels were measuredby EISA, following manufacturer's instructions (Cat No. DY1070 R&DSystems, Minneapolis Minn., USA). The table following table shows TNFαvalues of M2 macrophage cultures stimulated with TREM-1 ligand(PGLYRP1+PGN) in the presence of control antibody or anti-TREM-1antibody.

Donor 1 Donor 2 TNF-α, pg/ml TNF-α, pg/ml M2 macrophages with: Avg SDAvg SD Cells only 0.5 19.0 12.0 PGRP-S only 3.5 13.0 6.3 PGN BS only 5.72.8 22.4 3.8 PGN + PGRP-S 295.3 49.3 307.4 75.8 +Fc4 neg −3 248.5 54.0285.0 57.6 +Fc4 neg −1 215.6 41.5 295.0 59.0 +Fc4 neg −.33 217.5 49.7357.7 118.6 +Fc4 neg −.11 193.9 40.4 348.4 75.7 +Fc4 neg −.037 172.657.6 370.7 88.6 +Fc4 neg −.012 177.6 38.1 286.7 71.5 +Fc4 neg −.0041275.4 67.9 253.3 45.0 +0170-3 33.3 8.8 57.2 3.2 +0170-1 41.3 8.5 47.010.5 +0170-.33 45.3 14.3 80.4 21.6 +0170-.11 42.2 12.4 103.8 32.8+0170-.037 54.4 18.5 142.3 43.5 +0170-.012 86.2 24.4 203.8 55.6+0170-.0041 186.7 21.0 249.3 92.5 +0170-.0014 231.2 56.0 286.1 112.8+0170-.00046 246.4 22.7 254.2 70.5

This example illustrates that the anti TREM-1 Ab-0170 can effectivelyblock TREM dependent cytokine production in macrophage cells derivedfrom cynomolgus monkeys. The efficacy of this Ab supports its use incynomolgus monkey in in vivo toxicology and disease treatment models.

Example 19: TNFalpha Release from Stimulated Peripheral BloodMononuclear Cells can be Blocked by TREM-1 Antibodies

PBMC's, from a buffy coat and frozen in RPMI 1640 (Cat. no. 61870,Gibco, New York, USA), 20% FBS (Cat #16140-071, Gibco, New York, USA,10% DMSO (Cat #D2650, Sigma, Steinheim, Germany), were thawed and washedtwice in RPMI, 10% FBS, 1% Pen/Strep (Cat. no. 15070-06, Gibco, NewYork, USA), and resuspended in same medium to 4×10E6/ml. Cells were thendistributed with 400.000 cells/well. 10 μg/ml PGN-SA (Cat #tlrl-pgnsa,Invivogen, San Diego, USA) and 0.2 μg/ml PGLYRP1 were added to the wellsfor stimulating the cells. Subsequently, the relevant isotype and TREM-1antibodies were diluted in RPMI and added at 1.34 nM, and 0.167 nMrespectively. TNFalpha release were measured by diaplex (Cat#880.090.001, Genprobe, Besancon, France) according to manufacturersprotocol after 20 hrs incubation at 37° C., 5% CO₂. As shown in Table13, below, the TREM-1 antibodies disclosed herein (mAbs 0044, 0070 and0059) are all able to decrease the TNFalpha release from PBMC cells.

TABLE 15 % Inhibition compared to isotype Isotype at 1.34 nM 0 nM 0.167nM a-Trem-1 0044 (mIgG2a) 100 100 68 a-Trem-1 0070 (hIgG4) 100 117 76a-Trem-1 0059 (hzIgG1.1) 100 105 61

The approximately 70% inhibition of TNFalpha release in PBMCs using ablocking TREM-1 antibody indicates a significant impact on cytokinelevels in a stimulated cell culture.

Example 20: Anti-TREM-1 mAb can Inhibit PGN+PGLYRP1-Induced TNFαProduction in Normoxic Macrophages

Stimulation of macrophages using PGN-BS+human PGLYRP1 as a stimulant ofthe TREM-1 receptor can be blocked by anti-TREM-1 antibodies.

Monocytes were differentiated into M2 macrophages as in Example 15. Allsteps of the differentiation and stimulation of the cells were done in a37° C., 5% CO2 incubator under normal atmospheric oxygen levels(normoxia). The differentiated M2 macrophages were resuspended inRPMI/10% FBS and plated out at 5×10E5 cells/ml in triplicate (unlessotherwise noted) test wells. The cells were then stimulated for 24 hourswith the following stimulations: no addition. PGLYRP1, PGN-BS(InVivogen, tlrl-pgnbs)(two sets of triplicates), PGN-BS+PGLYRP1 (threesets of triplicates), or PGN-BS+PGLYRP1 in the presence of anti-TREM-1or isotype control antibody. Supernatants were then harvested andanalysed for TNFα using BioPlex (Bio-Rad, 171-85026M). Antibodies (0.1μg/mi) mAb-0122 and -0170 directed against TREM-1 were able to lower theTNF-alpha release.

TABLE 16 Donor 1 Donor 2 TNFa pg/ml TNFa pg/ml Macrophages stimulatedwith: Avg SD Avg SD No addition 0 0 0 0 PGLYRP1 0 0 0 0 PGN-BS 427 86381 85 PGN-BS + PGLYRP1 728 293 771 220 PGN-BS + PGLYRP1 + hIgG4 987 183884 41 isotype control PGN-BS + PGLYRP1 + mAb 243 14 367 16 0122PGN-BS + PGLYRP1 + mAb 122 106 224 54 0170

This example illustrates that anti-TREM-1 mAb-0122 and -0170 can inhibitTNFa production from macrophages grown under normoxic conditions.

Example 21: TREM-1 Antibody Specifically Inhibits Rheumatoid ArthritisSynovial Fluid-Induced Response

The RA synovial fluid samples from patients suffering from rheumatoidarthritis were assayed for TREM-1 ligand activity in the BWZ reporterassay as described in Example 6. Briefly, synovial fluid was thawed,vortexed, and serially diluted, assayed in duplicate +/− 10 μg/mlPGNECndi (Invivogen SanDiego, Calif., USA) with the addition of TREM-1antibodies or a negative isotype control. The synovial fluid from arheumatoid arthritis patient is able to trigger the BWZ/hTREM-1 reportercell assay in a TREM-1 dependent manner which is further enhanced byadding MAB1278 (R&D Systems, Minneapolis, Minn. 55413, USA: Cat. no.MAB1278)) whereas the blocking TREM-1 antibodies disclosed herein areable to decrease this activation.

The antibodies were tested in two assays indicated by the two columnsbelow, each antibody in concentrations ranging from 0.1 to 10 ug/ml.MAB1278 clearly enhanced the signal, whereas mAbs 0122 and 0170decreased the signal compared to the isotype control.

TABLE 17 + IgG4 isotype Concentration + mAb 0122 + mAb 0170 + mAb 1278control  10 ug/ml 80877 79731 52064 50304 1079000 1054000 306906 397556  1 ug/ml 74191 66458 48754 46978 1192000 1023000 398431 363267 0.1ug/ml 133900 163521 246695 169483 828866 691379 293831 313445

Example 22: Cytokine Release from Synovial Tissue Cells from RheumatoidArthritis Patients Upon Stimulation with PGLYRP-1 can be Blocked by mAb0170

Synovial tissue samples were obtained from RA patients during total kneereplacement. Single suspension of synovial tissue cells was isolated bya digestion via 4 mg/ml of collagenase (cat #11088793001, Roche,Mannheim, Germany) and 0.1 mg/ml of DNase (cat #11284932001, Roche,Mannheim, Germany) for 1 h at 37 degrees C.

Synovial tissue cells at 1×10{circumflex over ( )}5/well in culturemedium RPMI (cat #R0883, St Louis, Mo., USA)+10% FCS (cat #S0115,BioChrom AG, Grand Island, N.Y. 14072, USA) were stimulated with 4 ug/mlof PGLYRP1 and 1 ug/ml of PGN-ECNDi (cat #tlrl-kipgn, Invivogen, SanDiego, Calif. 92121, USA) under hypoxic condition in the presence orabsence of various concentrations of mAb 0170 or an isotype hIgG4control. After 24 h incubation, cell supernatants were harvested, andcytokines were measured by either ELISA (TNFa (cat #DY210, R&D,Minneapolis, Minn. 55413 USA), IL-1b (88-7010-88, eBioscience, SanDiego, Calif. 92121, USA), GM-CSF (cat #88-7339-88, eBioscience)) orFlowcytomix (TNFa, IL-1b, MIP-1b, MCP-1, IL-6, and IL-8 (cat #BMS,eBioscience). The cytokines were secreted from the synovial tissue cellsupon stimulation with the TREM-1 ligand and specifically blocked byTREM-1 antibody mAb 0170.

Below is an example of such experiment, where either 4 ng/ml or 10 ng/mlmAb was used resulting in a decrease of cytokine release when treatedwith TREM-1 antibody mAb 0170.

TABLE 18 Cytokine PGN + PGN + PGLYRP1 + 4 PGN + PGLYRP1 + (pg/ml) PGNPGLYRP1 10 ng/ml control 10 ng/ml mAb 0170 TNFalpha 624 1445 1034 429MIP- 2458 4395 3791 2321 1beta MCP-1 273 471 391 210

TABLE 19 Cytokine PGN + PGN + PGLYRP1 + 4 PCN + PGLYRP1 + (pg/ml) PGNPGLYRP1 ng/ml control 4 ng/ml 0170 IL-1beta 2419 3773 3477 2308 GM-CSF182 616 656 431 IL-6 2057 4189 3475 1632 IL-8 2575 5509 4499 2112

This example shows that cells from synovial tissue from rheumatoidarthritis patients will respond to stimulation by the TREM-1 ligand,PGLYRP1, by secreting numerous cytokines which can be inhibited by mAb0170.

Example 23: Type II PGLYRP1 Induced TNFalpha Release in Synovial TissueCells from Rheumatoid Arthritis Patients can be Blocked by TREM-1Antibody mAb 0170

Synovial tissue samples were obtained from RA patients during total kneereplacement. Single suspension of synovial tissue cells was isolated bya digestion via 4 mg/ml of collagenase (cat. no. 11088793001, Roche,Mannheim, Germany) and 0.1 mg/m of DNase (cat. no. 11284932001 Roche,Mannheim, Germany) for 1 h at 37 degree. The synovial tissue cells(1×10{circumflex over ( )}5/well in culture medium RPMI (cat. no.22400105, Gibco, NY 14072, USA)+10% FCS (cat. no. S0115, BioChrom AG,Berlin, Germany)) were co-cultured with various doses of HEK cellstransiently transfected with type II PGLYRP1 under hypoxic condition inthe presence or absence of 1 ug/ml of mAb 0170 or IgG4 isotype control.After 24 h incubation, cell supernatants were harvested, and cytokineswere measured by TNFa ELISA (cat. no. DY210, R&D, Minneapolis, Minn.55413 USA).

TABLE 20 Type II PGLYRP1 (HEK TNF-a (pg/ml) release transfected)/ControlHEK 1 × 10{circumflex over ( )}5 3 × 10{circumflex over ( )}4 1 ×10{circumflex over ( )}4 3 × 10{circumflex over ( )}3 3 × 10{circumflexover ( )}3 0 IgG4 + Type II cells 121.17 114.08 95.02 54.56 57.87 33.47IgG4 + Control cells 55.65 63.73 57.99 33.78 36.40 36.32 mAb 0170 + TypeII cells 44.05 44.67 44.40 39.45 44.29 31.40 mAb 0170 + Control cells54.50 57.06 53.10 42.10 31.99 27.82

This example shows that the TREM-1 ligand (type II cells) inducedTNF-alpha release in a dose-dependent manner in synovial tissue cellsfrom rheumatoid arthritis patients compared to control cells (mocktransfected). This TNFalpha response was blocked by mAb 0170 but notwith isotype IgG4. The control cells were not affected.

Example 24: Platebound MAB1278 Induced IL-6 and TNFalpha Response inMacrophages, Showing Agonistic Features, Whereas mAbs 0122 and 0170 Didnot

Stimulation of macrophages on platebound agonistic anti-TREM-1antibodies induced production of IL-6 and TNFa. Monocytes were purifiedfrom healthy donor buffy coats using RosetteSep (StemCell Technologies,15068) and differentiated into macrophages by culturing for 6 days inRPMI/10% FBS in the presence of 40 ng/ml human MCSF. The macrophageswere then further differentiated to M2 macrophages by changing the mediato growth media supplemented with 50 ng/ml human IL-4 and returning tothe incubator for an additional 24 hours. On the seventh day,macrophages were recovered from the culture plates by washing with1×PBS, followed by 5 mM EDTA in PBS. The plates were then returned to37° C. for 30 minutes before the macrophages were washed off the plates.The macrophages were washed in RPM/10% FBS before resuspending andplating out. The test wells had been pre-coated with the specifiedantibodies by incubating them overnight with antibody diluted in PBS,followed by washing ×3 in PBS. The resuspended macrophages were platedout at 5×10E5 cells/ml in triplicate test wells followed by incubationfor 24 hours. (All steps of the differentiation and stimulation of thecells were done in a 37° C., 5% CO2 incubator under normal atmosphericoxygen levels (normoxia)). Supernatants were then harvested and analysedfor IL-6 and TNFa using BioPlex (Bio-Rad, 171-B5006M and 171-B5026M).

Antibodies mAb-0122 and -0170 showed very low agonism whereas theMAB1278 antibody (RnD Systems, MAB1278) showed potent induction of bothIL-6 and TNFa.

TABLE 21 IL-6 pg/ml TNFa pg/ml Platebound mAb stimulation: Avg SD Avg SDNo antibody 1 3 0.3 0.8 mIgG1 isotype cntr 2 μg/ml 0 0 0 0 mIgG1 isotypecntr 6 μg/ml 0 0 0 0 mIgG1 isotype cntr 20 μg/ml 14 24 5 5 MAB1278 2μg/ml 412 71 2004 451 MAR1278 6 μg/ml 877 38 6454 278 MAB1278 20 μg/ml1352 76 7753 555 hIgG4 isotype cntr 2 μg/ml 5 5 2 1 hIgG4 isotype cntr 6μg/ml 18 15 9 9 hIgG4 isotype cntr 20 μg/ml 37 3 34 1 mAb 0122 2 μg/ml93 48 59 18 mAb 0122 6 μg/ml 126 8 105 17 mAb 0122 20 μg/ml 234 10 30426 mAb 0170 2 μg/ml 137 29 76 21 mAb 0170 6 μg/ml 181 12 187 28 mAb 017020 μg/ml 277 15 629 108

This example illustrates that mAb-0122 and -0170 only show very lowagonistic activity in macrophages and indicates true blocking featuresof these mAbs.

Example 25: Blocking TREM-1 in a Mouse Arthritis Model Reduces Disease

The experiments outlined in Table 22 were obtained in the DTH-arthritismodel, which is a single paw arthritis model. Single paw arthritis wasinduced in female C57BL/6 mice by eliciting a classical delayed-typehypersensitivity (DTH) reaction in the right hind paw by immunisationand subsequent challenge with methylated bovine serum albumin (mBSA),with the modification that a cocktail of type II collagen monoclonalantibodies (anti-C) was administered IV between the immunisation andchallenge steps. The left hind paw received PBS challenge and functionedas an intra-animal control. Mice (10 mice/group) were treated 3times/week with a TREM monoclonal antibody that specifically binds andblocks murine TREM-1, as determined using a murine version of thereporter assay described in Example 6. The first dose was administeredon the day of immunization. Mice (9-10 mice/group) were treated witheither a control antibody or PBS as a control. Paw swelling was measuredfrom the day of arthritis induction and 11 days onwards. Results arepresented as a mean area under the curve (AUC)±SEM. Statisticalsignificance was tested by using a two sided unpaired t-test, 95%confidence interval.

TABLE 22 Effect of TREM-1 treatment on paw swelling (AUC-mm) in a mousearthritis model. Effect Experi- TREM-1 Control measure ment mAb^(§)mAb^(§) PBS AUC-mm #1 8.03 +/− 1.01** 12.26 +/− 0.84 12.53 +/− 0.87 pawswelling AUC-mm #2 8.45 +/− 0.94** 11.71 +/− 0.22 12.89 +/− 0.36 pawswelling Means +/− SEM. ^(§)mice treated with 5 mg/kg, 3 times/week for3 weeks **P ≤ 0.005, two sided unpaired t-test, 95% confidence intervalvs. control mAb and vs. PBS

Example 26: Activated Neutrophils Release IL-8 which can be Blocked byTREM-1 mAbs

Neutrophils express TREM-1 and neutrophils also express the TREM-1ligand. To test whether TREM-1 is involved in an autocrine stimulationloop in neutrophils, isolated neutrophils were stimulated with PGN-SA(InVivogen, tlrl-pgnsa), and the release of IL-8 into the culture mediumwas measured. TREM-1 antibodies mAb-0059, -0067, -0122, and -0170 wereable to decrease the PGN-SA-induced IL-8 release. Neutrophils wereisolated from human healthy donor whole blood and resuspended inRPMI/10% FBS at. 5×10E6 cells/ml, and plated out into triplicate testwells pre-coated with Fibrinogen (pre-coated with 50□l of 1 mg/mlFibrinogen (Sigma, F3879) in PBS for 2 hours at 37° C., followed bywashing ×3 in PBS). The cells were tested under the followingconditions: no added stimulation, 10□g/ml PGN-SA only, or 10 □g/mlPGN-SA in the presence of mAb-0059, -0067, -0122, -0170 or isotypecontrol antibody at 0.25□g/ml. The samples were cultured 24 hours in a37° C., 5% CO₂ incubator. Supernatants were then harvested and analysedfor IL-8 using the Bio-plex Pro Human Cytokine IL-8 set (BioRad,171-B5008M).

TABLE 23 Experiment 1 Experiment 2 Neutrophils IL-8, pg/ml NeutrophilsIL-8, pg/ml stimulated with: Avg SD stimulated with: Avg SD No additionN.D. N.D. No addition 857 214 PGN-SA 562 54 PGN-SA 6116 191 PGN-SA +hIgG1.1 461 80 PGN-SA + hIgG4 6530 1962 isotype contr. isotype contr.PGN-SA + mAb 0059 165 55 PGN-SA + mAb 0122 2466 437 PGN-SA + mAb 0067183 77 PGN-SA + mAb 0170 2171 480

This example illustrates that IL-8 release from neutrophils induced bystimulation with the bacterially derived PGN can be reduced by TREM-1antibodies. Thus demonstrating that TREM-1 is involved in an autrocrineactivation loop in neutrophils, and the TREM-1 antibodies arepotentially useful in downregulating neutrophil responses.

Example 27: Activated Neutrophils can Stimulate Monocytes, which can beBlocked by Anti-TREM-1 mAbs

Activated neutrophils express the TREM-1 ligand. To test if activatedneutrophils can stimulate other immune cells in a TREM-1-dependentmanner, activated neutrophils were used to stimulate isolated monocytesand the release of TNFα into the culture medium was measured. TREM-1antibodies mAb-0059 and -0170 were able to decrease theneutrophil-induced TNFα release from the monocytes.

Neutrophils were isolated from human healthy donor whole blood andresuspended in RPMI/10% FBS, and plated at 1.5×10E5 cells/well inpoly-D-Lysine coated tissue culture 96-well plates (Corning, 3841). Theneutrophils were then stimulated with 1 ng/ml PMA (Sigma, P1585)+20□g/mlPGN-SA (InVivogen, tlrl-pgnsa) for 24 hours in a 37° C., 5% CO2incubator. The cells were then washed gently ×3 with media before addingin freshly isolated monocytes. The monocytes were purified from healthydonor buffy coats using an EasySep kit (Stem cell technologies, 19059),and were plated out with 5×10E4 cells/well in the wells alreadycontaining activated, washed neutrophils. The following antibodies wereadded at 1 g/ml: mAb-0059, mAb-0170, or hIgG4 isotype control. The cellswere then cultured for another 24 hours before harvesting thesupernatant. The supernatant was diluted 1:10 in RPMI/10% FNS beforemeasuring TNFa by ELISA (eBioscience, BMS223INST).

TABLE 24 TNFa pg/ml Monocytes stimulated with: Avg SD No neutrophils 3485 Activated neutrophils 299 67 Activated neutrophils + isotype control232 32 Activated neutrophils + mAb 0059 72 14 Activated neutrophils +mAb 0170 129 9

This example illustrated that activated neutrophils can stimulatemonocytes in a TREM-1-dependent manner to produce TNFa, and this can beblocked by mAb-0059 and mAb-0170 anti-TREM-1 antibodies. Anti-TREM-1antibodies are therefore potentially useful for downregulating monocyteresponses.

Example 28: Epitope Mapping by Protein Crystallography Materials

The IgV-like domain of human TREM-1 (SEQ ID NO: 24) in a bufferconsisting of 20 mM 2-(N-morpholino)ethanesulfonic acid, 150 mM NaCl, 5%(v/v) glycerol, pH 5.5 at a protein concentration of 11.7 mg/mL

The Fab region of mAb 0170 (SEQ ID NO: 25 and SEQ ID NO: 26) in a bufferconsisting of 10 mM phosphate, 2.68 mM KCl, 140 mM NaCl, pH 7.4 at aprotein concentration of 8.5 mg/mL.

Methods: Protein Complex Formation and Crystallization

The IgV-like domain of human TREM-1 was mixed with the Fab region of mAb0170 in their original buffers in a 1:1 molar ratio, giving a finalconcentration of Fab of 7.1 mg/mL. The proteins were co-crystallized ina hanging drop vapour diffusion experiment by equilibration of dropletsconsisting of 2 μL protein solution mixed with 2 μL reservoir solutionagainst a 0.5 mL reservoir composed of 0.01 M MgCl₂, 0.005 M NiCl₂, 0.1M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 13 (w/v) %polyethylene glycol 3350, pH 7.0. The crystals appeared as clusters ofcrystals, which were separated into single crystals and transferred to adrop consisting of 35 (w/v) % polyethylene glycol 3350, 0.01 M MgCl₂,0.005 M NiCl₂ and left to equilibrate in the drop for 15 seconds. Thecrystal was mounted in a 0.2 mm diameter litholoop (Molecular DimensionsLimited) and flash-cooled in liquid nitrogen.

X-Ray Diffraction Data Collection, Structure Determination andRefinement

Diffraction data were collected at a micromax-007HF Cu X-ray generator(Rigaku Europe) operated at 40 kV, 30 mAmp and equipped with Varimax HFoptics (Cu K□, =0.15418 nm)(Rigaku Europe), a Cryonix165 CCD (RigakuEurope) and a Cryo-stream (Rigaku Europe) operated at 100 K. The rawdata images were indexed, integrated and scaled using the XDS programpackage (Kabsch, Acta Crystallogr. D66, 133-144 (2010)). The space groupof the crystal was P2(1), with unit cell parameters, a=62.3 Å, b=64.9 Å,c=74.4 Å, α=90°, β=75.2°, γ=90°. Data were collected to a resolution of1.99 Å. The structure was solved by molecular replacement using thePhenix software (Adams et al., Acta Crystallogr. D66, 213-221 (2010)) asimplemented in the CCP4i program suite (Potterton et al., ActaCrystallogr. D59, 1131-1137 (2003)). The search models were thestructure of the mAb 0170 Fab fragment and the IgV-like domain fromhuman TREM-1 (Kelker et al., J. Mol. Biol. 342, 1237-1248 (2004)). Onecopy of each molecule was located in the asymmetric unit. Structurerefinement was carried out using Refmac5 (Murshudov er al., ActaCrystallogr. D53, 240-255 (1997)) from the CCP4i program suite. Cootversion 7 (Emsley et al., Acta Crystallogr. D66, 486-501 (2010)) wasused for manual structure rebuilding and validation.

Results and Discussion

The crystal structure of the complex between the Fab fragment of mAb0170 and the IgV-like domain of human TREM-1 included E1-V219 (SEQ IDNO: 25), D1-C218 (SEQ ID NO: 26) and L24-T134 of SEQ ID NO: 24. Thequality parameters of the structure were good with overall R-factor ofthe structure=21.1% and the Free R-factor=; 27.1%. The overallcorrelation coefficient was 0.93 and the diffraction-component precisionindex, DPI=0.2 Å (Cruickshank, Acta Crystallogr. D55, 583-601 (1999)).The root-mean-square deviation of the bond lengths in the structure fromideal bond lengths=0.0164 Å and the root-mean-square deviation fromideal bond angles=1.8661° (Engh and Huber, Acta Crystallogr. A47,392-400 (1991)). Regions displaying intermolecular distances of lessthan or equal to 4 Å between the IgV-like domain of human TREM-1 and theFab fragment of mAb 0170 were assigned to the epitope region of humanTREM-1 (SEQ ID NO: 1). The analysis of intermolecular distances wascarried out using the program NCONT in the CCP4 program suite (Pottertonet al., Acta Crystallogr. D59, 1131-1137 (2003)). The analysis showedthat human TREM-1 amino acid residues K40, D42, T44-K47 and Y90-L95, R97defined the epitope.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now be apparent to those of ordinary skill in the art.It is, therefore, to be understood that the appended claims are intendedto cover all such modifications and changes as fall within the truespirit of the invention.

1. An isolated nucleic acid encoding an antibody or fragment thereofwhich binds to a triggering receptor expressed on myeloid cells-1(TREM-1) having an amino acid sequence set forth as SEQ ID NO: 1 (humanTREM-1), SEQ ID NO: 12 or SEQ ID NO: 21 (cynomolgus monkey TREM-1),wherein the antibody or fragment thereof comprises a heavy chainvariable region that comprises CDR1, CDR2, and CDR3 domains; and a lightchain variable region that comprises CDR1, CDR2, and CDR3 domains, andwherein the heavy chain variable region CDR3 domain comprises amino acidresidues 101 to 110 of SEQ ID NO:
 4. 2. The nucleic acid of claim 1,wherein the antibody or fragment thereof specifically binds to D38 toF48 of SEQ ID NO:
 1. 3. The nucleic acid of claim 2, wherein the heavychain variable region CDR2 domain comprises amino acid residues 50 to 68of SEQ ID NO: 4 or amino acid residues 50 to 88 of SEQ ID NO: 4 withone, two, or three amino acid substitutions.
 4. The nucleic acid ofclaim 2, wherein the heavy chain variable region CDR1 domain comprisesamino acid residues 31 to 35 of SEQ ID NO: 4 or amino acid residues 31to 35 of SEQ ID NO: 4 with one amino acid substitution.
 5. The nucleicacid of claim 2, wherein the light chain variable region CDR3 domaincomprises amino acid residues 93 to 101 of SEQ ID NO: 5 or amino acidresidues 93 to 101 of SEQ ID NO: 5 with one or two amino acidsubstitutions.
 6. The nucleic acid of claim 2, wherein the light chainvariable region CDR2 domain comprises amino acid residues 54 to 60 ofSEQ ID NO: 5 or amino acid residues 54 to 60 of SEQ ID NO: 5 with one ortwo amino acid substitutions.
 7. The nucleic acid of claim 2, whereinthe light chain variable region CDR1 domain comprises amino acidresidues 24 to 38 of SEQ ID NO: 5 or amino acid residues 24 to 38 of SEQID NO: 5 with one, two, or three amino acid substitutions.
 8. Thenucleic acid of claim 2, wherein the heavy chain variable region CDR1domain comprises amino acid residues 31 to 35 of SEQ ID NO: 4 andwherein the heavy chain variable region CDR2 domain comprises amino acidresidues 50 to 68 of SEQ ID NO:
 4. 9. The nucleic acid of claim 2,wherein the light chain variable region CDR3 domain comprises amino acidresidues 93 to 101 of SEQ ID NO: 5 with one or two amino acidsubstitutions.
 10. The nucleic acid of claim 1, wherein the antibody orfragment thereof binds human TREM-1 with a binding affinity (K_(D)) thatis or 1×10⁴M or less, as determined using surface plasmon resonance. 11.The nucleic acid of claim 1, wherein the antibody or fragment thereoffurther comprises a heavy chain constant region which is of a humanIgG1, IgG2, IgG3, or IgG4 isotype.
 12. The nucleic acid of claim 1,wherein the antibody or fragment thereof further comprises a heavy chainconstant region which is of a human IgG1 or IgG4 isotype.
 13. Thenucleic acid of claim 1, wherein the fragment is a Fab, Fab′, F(ab′)₂,dAb, Fd, Fv, a single chain Fv fragment, or an isolated CDR. 14.(canceled)
 15. An isolated nucleic acid encoding an antibody or fragmentthereof which competes with mAb 0170 for binding to a triggeringreceptor expressed on myeloid cells-1 (TREM-1) having an amino acidsequence set forth as SEQ ID NO:1 (human TREM-1), SEQ ID NO:12 or SEQ IDNO:21 (cynomolgus monkey TREM-1).
 16. A vector comprising the nucleicacid of claim
 1. 17. A cell comprising the nucleic acid of claim
 1. 18.A method of making an antibody or fragment thereof which binds to atriggering receptor expressed on myeloid cells-1 (TREM-1), comprisingexpressing the antibody in the cell of claim 17.